Accurate atomic resolution XFEL structures of a metalloenzyme reveal key insights into its catalytic mechanism*

Domain: chemistry
Published: 2026-03-07
Editorial outcome: major_revision

Abstract
Metalloproteins represent a major fraction of the protein kingdom and often exploit redox chemistry of
transition metals to drive key biological events involving proton and electron transfer. Copper is one of
the most widely used transition metals whose redox properties are utilised in both electron transfer and
catalysis of chemical substrates. Copper nitrite reductases (CuNiRs) utilise two types of copper centres
and have become a model system for studying complex biological events that underpin the reaction
mechanisms of redox enzymes, including proton-coupled electron transfer and substrate gating. We
utilises the higher X-ray energy (13 keV) available at the SACLA X-ray Free Electron Laser (XFEL)
and SHELXL refinement to obtain accurate atomic resolution structures of CuNiRs at ~1 Å from three
organisms – in the oxidised (low and high pH), reduced and substrate-bound states. A consistent picture
now emerges with the observation of a pentacoordinated oxidisEed catalytic type-2 Cu (T2Cu2+) centre in
all cases. A tetracoordinated reduced T2Cu+ site with a single solvent ligand has also been captured
giving structural support to the random-sequential sche me.

Introduction
Copper is one of the most commonly occurring redox-active metals in enzymes and plays a significant
role in chemical biology. Its diverse redox-active chemical reactivity enables it to participate in various
complex biological processes. Its ability to change reversibly between the Cu(II)/Cu(I) redox state
during catalysis and electron transfer processes, while remaining coordinated to the amino acid ligands,
gives it versatility1. These chemical properties are harnessed in a wide variety of oxidoreductases
including cytochrome c oxidase2, superoxide dismutase3, tyrosinase4, oxygenases5 and nitrogen oxide (NO ) reductases6.
x

Copper nitrite reductases (CuNiRs) catalyse the reduction of nitrite (NO -) to nitric oxide (NO) and are a
2
key enzyme in the denitrification step of the nitrogen cycle6,7 . EThe redox reaction involves two protons,
one electron and the chemical substrate nitrite: NO 2 - + e- + 2H+  NO + H 2 O. Two distinct classes of
nitrite reductases are found in denitrifying organisms, a cytochrome cd
1
type, encoded by nirS and the
more widely distributed copper-containing enzymes encoded by nirK6,7. The Cu-containing nitrite
reductases have been extensively studied and s hown to be a trimer with each monomer having two
domains with a characteristic -sandwichL motif8,9. Each monomer contains a type-1 Cu (T1Cu) centre
close to the protein surface and a type-2 Cu (T2Cu) site at the interface between two monomers (Fig. 1).
In the oxidized enzyme the T1CuT shows strong absorbance bands in the visible region and variation in
their intensity results in the colour varying from blue to green depending on the organism10. The T1Cu
centre accepts electrons from either a cytochrome c or a cupredoxin and are transferred to the T2Cu
centre where the chemical reaction for the conversion of NO - to NO occurs11. Two invariant residues,
2
catalytic Aspartic acid residue (Asp ) and catalytic Histidine residue (His ), located in the active
CAT CAT
site pocket have a proposed role in proton delivery during catalysis, and an Ile residue provides steric
CAT
modulation of ligand binding to the T2Cu12,13. Asp is mobile and has been observed in three different
CAT
positions termed “gatekeeper” (pointed away from the T2Cu), “proximal” (orientated towards the T2Cu
and H-bonded to a liganded H O) and distorted proximal where the carboxyl Oδ2 atom is tilted closer
2
towards a T2Cu ligand14–16. Nitrite binds to the T2Cu by displacement of a H O ligand forming an
2
adduct that shows some variation in symmetry and “top hat” and “side on” conformations14–17. The two
Cu centres, separated by 12.5 Å, are directly linked via a unique Cys-His bridge formed by
neighbouring residues that are ligands to the two Cu centres. Both copper centres cycle reversibly
between the Cu(II)/Cu(I) redox state during catalysis, a feature in common with redox centres of many
key metalloenzymes such as cytochrome c oxidase2, hydrogenases18,19, nitrogenases20,21 and nitrite
reductases7. In these systems, as with CuNiRs, catalysis involves the controlled delivery of electrons
and protons to the active site where chemical substrates are utilised. The amenability of CuNiRs for
biophysical study and highly diffracting crystals capable of providing high resolution crystallographic
structures, have made them an attractive model system for studying these processes with a view of
establishing general principles that underpin catalytic redox reactions in these systems.

In CuNiRs, it has been demonstrated that inter-Cu electron transfer (ET) is proton-coupled and during
catalysis it is gated by nitrite binding to the catalytic T2Cu centre22–24. Recent high resolution MSOX
(multiple structures from one crystal) studies, combined with on-line single crystal spectroscopy during
substrate-free and substrate-bound states, has provided evidence for the electronic route of proton-
coupled electron transfer (PCET) from T1Cu to T2Cu (S() E T1Cu+→ T2Cu2+)2528.
Cys

In general, redox chemical reactions during biological catalysis are often accompanied by only subtle
changes (< 0.1 Å in bond length) in the coordination sphere which can only be determined at very high
resolutions approaching atomic resolution ~1.2 Å. Such resolutions have been achieved using
synchrotron radiation-based crystallograpLhy for CuNiRs14,16,26. However, the X-rays from synchrotron
sources used to obtain high resolution structures themselves introduce redox activation of these enzymes
in the crystals during a typical crTystallographic data collection. The photoreduction of the T1Cu centre
in crystals results in the bleaching of the T1Cu optical spectrum, while in the nitrite (substrate) bound
crystals it results in the conversion of nitrite to NO alongside the bleaching of the optical
spectrum22,25,27,28. This serious limitation of unintended redox changes has been overcome by the use of
X-rays from Free Electron Lasers (XFEL) that has allowed single shot experiments using femtosecond
pulses of X-rays. This enables time-frozen (~10 fs) structures to be obtained that are essentially free
from radiation-induced chemistry (FRIC structures). Both XFEL based crystallographic approaches,
namely serial femtosecond crystallography (SFX) and serial femtosecond rotational crystallography of a
single crystal (SF-ROX), have been used for CuNiRs. Single shot XFEL experiments have provided
much useful information on pure resting state and substrate bound states, but the resolution has
remained limited, -the highest resolution for an SF-ROX structure was 1.3 Å16 and for SFX was 1.6 Å17.
The recent availability of higher X-ray energy (<13 keV) at the SACLA XFEL has enabled structure
determination to atomic resolution <1.2 Å29 where refinement of anisotropic displacement parameters
(ADPs) and estimation of standard uncertainties (esds) through unrestrained refinement can be
performed in SHELXL30.

Here, atomic resolutions achieved using higher energy (13 keV) X-rays from an XFEL (Supplementary
Table 1) is combined with full SHELXL refinement (including one cycle of unrestrained refinement) for
refinement of ADPs and to estimate esds of bond lengths and angles around metal centre sites in a
metalloprotein system (CuNiR). This has been done for several important functionally relevant states
(low and high pH oxidised, substrate-bound and chemically reduced states). It has been possible to
collect data for many states in a limited allocated time using the recently implemented automatic data
collection pipeline for our SF-ROX data collection at SACLA. These structures offer unprecedented
accuracy and reveal several features of chemical importance in CuNiRs, namely the consistent presence
of two solvent molecules bound to the oxidised T2Cu(II) in a five-coordinated site for both the less
catalytically efficient bluish-green CuNiRs from BradyrhizobiuEm species (Bradyrhizobium sp. ORS
37531 or Bradyrhizobium japonicum32 - BrNiR) in their resting state and also the prototypic green
CuNiR from Achromobacter cycloclastes (AcNiR)14. T he mode of substrate binding for both also
indicates a consistent five-coordinated T2Cu(II) site with structural and spectroscopic evidence for the
strong preference of substrate binding to the ox idised T2Cu(II) before inter-Cu ET occurs. The
coordination geometry is altered upon redLuction to T2Cu(I). These observations collectively support
that the binding-before-reduction33 branch of the proposed “random sequential mechanism”34
predominates during turnover.

Results
Accurate atomic resolution XFEL structures of Bradyrhizobium CuNiRs
Our updated (automated high energy) SF-ROX data collection strategy for atomic resolution XFEL
structure determination was applied to obtain cryogenic (100 K) XFEL structures of Bradyrhizobium
CuNiRs (BrNiRs) from either Bradyrhizobium sp. ORS 375 or Bradyrhizobium japonicum at resolutions
of 1.00 Å to 1.15 Å - for as-isolated (fully-oxidised) states at pH 5.5 and pH 7.3, a nitrite-bound and
dithionite-reduced state, respectively (Supplementary Table 1,2). With the extension of the resolution
limits to ≤1.15 Å, unrestrained SHELXL refinement30 could now Salso be performed for these states
providing highly accurate bond distance and angle information. The current structures, together with
one cycle of unrestrained refinement for estimation of standard uncertainties (esds) [provided in
brackets], represent an example of redox enzyme syste ms, where XFEL structures, free from radiation
induced chemistry, are obtained for several key functional states at such high resolutions.

As-isolated oxidised BrNiR (pH 5.5) at L1.15 Å resolution. A common observation for these
Bradyrhizobium CuNiRs has been the presence of two water molecules coordinated to T2Cu in the
resting state active site, represent T ing a pentacoordinated architecture16,25,28. The atomic resolution XFEL
structure of as-isolated oxidised BrNiR pH 5.5, determined at 1.15 Å resolution, was obtained and again
revealed this coordination with two full occupancy water molecules seen bound to oxidised T2Cu(II)
(W1 and W2) (Fig. 2a,d) - an Fo-Fc OMIT map is also shown for clarification (Supplementary Fig. 1a)
However, in this structure the coordination and bond distances are slightly altered compared to the
equivalent cryogenic XFEL structure at 1.3 Å resolution16 (coordination distances are given in
Supplementary Table 3 and Supplementary Fig. 2a.). In the current structure, W2 is coordinated to the
T2Cu(II) at 2.08 (2) Å, similar to the lower resolution (1.3 Å) SF-ROX structure but W1 is at a
considerably shorter coordination distance of 1.83 (2) Å compared to 1.94 Å seen previously. The two
waters are also separated by shorter distance of 2.23 Å. The shortened W1-Cu(II) distance of 1.83 (2) Å
may indicate the presence of a hydroxide (OH-) ion ligated to T2Cu(II), which also hydrogen bonds to
the proximal position of the catalytic Aspartic residue (Asp92; Asp ), observed in this structure.
CAT
Asp also hydrogen bonds to an additional water (W3) in the active site that appears to be mobile.
CAT
These observations are qualitatively similar to the lower resolution XFEL structure, albeit W3 being
mobile as opposed to being well-defined. The catalytic Histidine residue (His250; His ) bridges
CAT
Asp through a conserved bridging water (W ) and its imidazole ring is rotated towards Glu274, with
CAT Br
its Nδ1 atom hydrogen bonding with the carbonyl oxygen of Glu274 at 2.70 Å (Supplementary Fig. 3a).
This is consistent with the previous SF-ROX structure at 1.3 Å resolution16.

The optical in crystallo spectrum of one of the crystals prior to XFEL collection confirmed the T1Cu
site to be fully oxidised (Fig. 3a). The atomic resolution XFEL structure of the oxidised crystal showed
a typical distorted tetrahedral geometry for T1Cu(II), with slight elongation of the Met-T1Cu(II)
coordination and shortening of Cys-T1Cu(II), more akin to a blue CuNiR compared to green (Fig. 2a
and Supplementary Fig 4a). The T1Cu(II) site shows no difference to the previous lower resolution-SF-
ROX structure (coordination distances are given in Supplementary Table 3 and Supplementary Fig 4a).

As-isolated oxidised BrNiR (pH 7.3) at 1.00 Å resolution. The catalytic activity of CuNiRs, including
the tethered CuNiRs with additional tethered redox dom ains, show a bell-shaped pH profile with an
activity maximum around pH 5.5-6.023,32,35–37. The bell-shaped profile is attributed to the involvement of
Asp and His in proton delivery. Here, in addition to pH 5.5, we have also undertaken XFEL
CAT CAT
structure determination of the as-isolated Loxidised BrNiR at high pH (pH 7.3) to 1.00 Å resolution
(Supplementary Table 1,2), This represents the only pH-dependent damage-free XFEL structure of any
CuNiR and shows that the generTal T2Cu(II) architecture is markedly different compared to the lower pH
structure.

The 1.00 Å resolution of this XFEL structure, together with SHELXL refinement, again suggests the
potential presence of a T2Cu(II) OH- ligand in this state instead of a water molecule. The T2Cu site
shows a highly ordered ligand (H O/OH-;W1) coordinated in a perfect tetrahedral geometry relative to
2
the histidine plane and a half-occupancy water (W2) in a position similar to W2 in the low pH structure
(Fig. 2b,e) - a Fo-Fc OMIT map is also shown for clarification (Supplementary Fig. 1b). The H O/OH-
2
ligand (W1) is tightly bonded to W2 by 2.16 Å with a relatively short W1-Cu coordination distance of
1.90 (1) Å. W2 has a longer T2Cu(II) coordination distance of 2.20 (2) Å (Supplementary Fig. 2b and
Supplementary 3). Unusually, the H O/OH- (W1) Cu ligand also appears to bridge between the two
2
catalytic residues – Asp and His , as opposed to the bridging water (W ) normally observed, with
CAT CAT Br
this bridging mode not observed before for any CuNiR. Favourable hydrogen bonds are formed with the
carboxyl oxygen (Oδ2) atom of Asp (2.55 Å) and the Nε2 atom of His (2.87 Å). In addition, a
CAT CAT
dual-occupancy partial water (W3) is also seen within the T2Cu active site pocket, providing some
linkage between W2 and Asp His is also favourably rotated towards Glu274 with a hydrogen
CAT. CAT
bonding distance of 2.71 Å (Supplementary Fig. 3b)

The coordination distances at the oxidised T1Cu(II) site are consistent with a fully oxidised species and
were confirmed by an optical in crystallo spectrum taken on a crystal prior to XFEL collection (Fig 3b.).
The T1Cu(II) Met145 ligand shows two positions, whilst maintaining coordination, with the distances
of 2.55 (1) Å (69 %) and 2.72 (1) Å (31 %), (Fig. 2b, Supplementary Table 3 and Supplementary Fig.
4b). The distance of the Met axial ligand has been associated with an increase in redox potential of the
T1Cu centre38,39. Other than the distortion of the T1Cu Met ligand, comparison with the lower pH
T1Cu(II) coordination reveals minimal differences (Supplementary Fig. 4h). Small differences are
reflected in the optical spectrum where the high pH spectrum sEhows ~ 2 nm shift of the 450 nm peak,
with the ratios between the two peak maxima also decreasing by ~ 10 % (Fig. 3a,b).

Nitrite-bound oxidised BrNiR (pH 5.5) at 1.02 Å resolution. There are a few atomic resolution
structures of nitrite bound CuNiR that have bee n obtained using synchrotron radiation (SR) X-rays. It is
now apparent that these synchrotron strucLtures are all affected to some extent by the X-ray radiolysis
during the data collection resulting in reduction of the T1Cu site and transfer of electrons to the nitrite
bound T2Cu site, initiating the enTzymatic turnover. These structures, thus, show mixed population of
substrate, sometimes in more than one conformation and the product NO. Single shot experiments on
CuNiRs have been performed using the femtosecond (~10 fs) X-ray pulses from XFEL to outrun the
radiation-induced chemistry. Both types of serial femtosecond crystallography have been used: SFX
which uses a large number of small (<5m) crystals and serial femtosecond rotational crystallography
(SF-ROX) which utilises fewer larger crystals where the crystal is translated between rotation. The best
resolution of nitrite bound structures of CuNiR using SFX and SF-ROX based has been ~1.6 Å17 and
1.3 Å16 respectively. We applied 13 keV X-rays from SACLA to obtain an atomic resolution structure
of oxidised nitrite-soaked BrNiR (pH 5.5), free from radiation-induced chemistry, at 1.02 Å resolution
(Supplementary Table 1,2). An optical spectrum of a nitrite-soaked crystal prior to collection of XFEL
data, clearly confirmed the presence of nitrite and the Cu(II) oxidation state of the T1Cu centre (Fig.
3b). The structure showed a fully occupied nitrite molecule coordinated to T2Cu(II) (Supplementary
Fig. 1c) with equal N-O bond lengths (~1.27 Å; O1-N [1.27 (3)]/O2-N [1.27 (4)], an angle of 131.5° (3))
and a binding mode resembling a “top-hat” conformation (Fig. 2c,f) - where the two oxygen atoms
(O1/O2) would be expected to be coordinated in a bidentate binding mode, with no interaction of the
nitrogen atom. However, in this structure, the coordinated nitrite molecule appears to be show
asymmetric bidentate binding (O2: 1.96 (2) Å, N: 2.27 (3) Å, O1: 2.25 (1) Å) (Supplementary Table 3
and Supplementary Fig. 2c) with the oxygen atom (O2) closest to Asp coordinating more favourably
CAT
to T2Cu(II) than the other two atoms. This observation contrasts with the previous lower resolution
structure where a “side-on” nitrite molecule was seen coordinated to the T2Cu(II) with almost
equidistant coordination (O2: 1.92 Å, N: 2.02 Å, O1: 2.12 Å)16. The structure also reveals the presence
of both the proximal conformation of Asp (83 %) and gatekeeper conformation (17 %) (Fig. 2c,f and
CAT
Supplementary Fig. 1c), which was not seen in the previous 1.3 Å resolution SF-ROX XFEL structure16.
The higher resolution of 1.02 Å has provided a significant improvement in the quality of map to resolve
the minor conformation. The gatekeeper position has been seen previously in the SR crystallographic
structures of BrNiR and other prototypic CuNiRs (i.e. AcNiR, AEfNiR) to varying degrees, but in these
cases multiple conformations of nitrite or nitrite and product are observed. The presence of gatekeeper
conformation with full occupancy single conformation of nitrite likely suggests that the “top-hat”
conformation of nitrite represents the initial conformation of the binding to T2Cu after delivery of nitrite
into the pocket. The proximal/gatekeeper positi ons of Asp are both present with new partial
CAT
occupancy waters near the active site cavLity: a gatekeeper water (W
G
; 14 %) which takes the place of
the second carboxyl oxygen (Oδ1) atom of Asp in the minor conformation and a proximal water (W ;
CAT p
20 %), which occupies a similar Tposition to W3 seen in the other structures, with the major
conformation. New mobile waters W4a/W4b are also observed closer to the nitrite molecule. The Oδ2
atom of the Asp proximal position bonds favourably with the O1 atom of the T2Cu-bound nitrite
CAT
molecule (2.34 Å) – this distance is almost identical to the W1-Oδ2 (Asp ) distance in the low pH
CAT
oxidised structure (2.36 Å). The conserved bridging water (W ), bridges again with Asp and His ,
Br CAT CAT
with His again rotated towards Glu274 with a hydrogen bonding distance of 2.63 Å (Supplementary
CAT
Fig. 3c). At the T1Cu(II) site, disorder of the Met145 T1Cu ligand is observed once more (Fig. 2c) but
with equal proportions this time (ordered (52%; 2.47 (7) Å); disordered (48%; 2.71 (8) Å))
(Supplementary Table 2 and Supplementary Fig. 4c).

Chemically reduced BrNiR (pH 5.5) at 1.05 Å resolution. A reduced form of any BrNiR has yet to be
structurally characterised and only a few structures in general of CuNiR have been reported for the
reduced enzymes15,40–43. A cryogenic XFEL structure of the reduced form of AcNiR was previously
obtained using the SF-ROX approach at 1.6 Å resolution15. To extend on that result and provide fresh
insight of the reduced form of a BrNiR, an atomic resolution XFEL structure of chemically reduced (by
dithionite) BrNiR at pH 5.5 was also successfully determined at 1.05 Å resolution, with each crystal
soaked with the strong reductant dithionite before cryocooling for SF-ROX data collection. The optical
spectrum of a crystal was obtained beforehand to ensure that full reduction of T1Cu(I) had occurred,
with full bleaching of T1Cu absorbance peaks observed (Fig. 3a).

At the reduced T1Cu(I) site, marked differences are observed when compared with the equivalent
oxidised form (atomic resolution XFEL structure of as-isolated BrNiR (pH 5.5) at 1.15 Å resolution). In
the reduced form of the enzyme, two positions of the T1Cu(I) are observed (Fig. 4d,e,f and
Supplementary Fig. 4d), representing two reduced states - with the major conformation (77 %) shifting
0.17 Å from its oxidised position (Supplementary Fig. 4g). This is similar to the equivalent AcNiR
structure15, where T1Cu shifted 0.2 Å upon reduction and also Erevealed two positions of the reduced
state of T1Cu. To our surprise though, in this higher resolution chemically-reduced BrNiR structure, the
major reduced T1Cu is associated with a classical tetra coordinated site (Fig. 4e and Supplementary Fig.
4d) but the minor reduced position (23 %) is associated with a unique tricoordinated site (Fig. 4f and
Supplementary Fig. 4d), where the Sδ atom of t he coordinating Met145 ligand is completely flipped
from the coordination sphere. This was pLreviously only observed in an as-isolated BrNiR enzyme at
high pH, following prolonged X-ray exposure of the same crystal volume in an MSOX experiment
using a focused X-ray beam at a Tsynchrotron beamline25.

At the reduced T2Cu(I) site, two active site conformations and positions of T2Cu(I) are observed,
representing a tetracoordinated T2Cu(I) site where a solvent ligand (Wa) is retained and a tricoordinated
T2Cu(I) site where the solvent ligand (Wa) is absent (Fig. 4a,b,c and Supplementary Figs. 1d and 2d).
The T2Cu drops 0.5 Å into the histidine plane in the major conformation (66 %), with the minor
position (34 %) similar to that seen in the other atomic resolution BrNiR XFEL structures also present.
In the active site cavity, a half-occupancy water ligand (Wa; 54 %) is observed coordinated to T2Cu in
one active site conformation (Fig. 4b and Supplementary Fig. 2d), with coordination distances of 1.72 Å
and 2.16 (2) Å for the two T2Cu(I) positions, respectively. In the second conformation, the Wa ligand is
lost and the site becomes devoid of an external ligand, with the Cδ1 side-chain of Ile252 flipping into the
available free space (46 %) (Fig. 4c and Supplementary Fig. 1d). The latter is consistent with a single
tricoordinated T2Cu(I) site that was observed in the lower resolution chemically reduced SF-ROX
structure of AcNiR at 1.6 Å resolution15. Asp remains in a single proximal position, with some
CAT
mobility of the Oδ2 atom observed and dual-occupancy water (W3) also present. His is instead
CAT
rotated towards the hydroxyl oxygen atom (Oϒ1) of Thr275, with roughly equal hydrogen bond
distances of 2.67 Å and 2.75 Å between the Glu274 and Thr275 residues, respectively (Supplementary
Fig. 3d).

Accurate atomic resolution XFEL structures of prototypic AcNiR
Cryogenic (100 K) XFEL structures of the prototypic AcNiR in its fully-oxidised as-isolated and nitrite-
bound catalytic states were also obtained at pH 4.8, both at resolutions of 0.95 Å (Supplementary Table
1,2). To the best of our knowledge, these sub-atomic (<1 Å) resolution structures represent the highest
resolution XFEL structures to date for a macromolecular molecule. The previous cryogenic XFEL
structures obtained for AcNiR using the SF-ROX approach were limited to a resolution of 1.5 Å15. With
the extension of the resolution to ≤1 Å, unrestrained SHELXL Erefinement could now also be performed
for both structures, providing highly accurate bond distance and angle information relevant to the redox
chemical reaction in these model biological systems (e sds values are provided in brackets).

As-isolated oxidised AcNiR (pH 4.8) at 0.95 Å resolution. We present the structure of as-isolated
oxidised AcNiR (pH 4.8) at a sub-atomic Llevel of resolution of 0.95 Å (Fig. 5). The structure revealed
previously unseen details of the T2Cu(II) site architecture that were not observable in the previous much
lower resolution (1.5 Å) SF-ROXT structure15. The higher resolution map has enabled the identification
of two distinct active site conformations, as seen in the Fo-Fc OMIT maps (Supplementary Fig. 1e): in
one, the fully occupied water molecule seen previously can again be observed in a distorted geometry
relative to the histidine plane (i.e in a position reminiscent of W2 denoted for Bradyrhizobium CuNiRs).
However, in this conformation, an additional partial T2Cu(II) water molecule (W1; 35 % occupancy) is
now also observed, revealing a fractional pentacoordinated oxidised state (Fig. 5c) for a prototypic
CuNiR (i.e. AcNiR, AxNiR and AfNiR). The partial water is positioned similarly to W1 of
Bradyrhizobium CuNiRs and is hydrogen bonded to W2 (2.32 Å) but is coordinated to T2Cu by an
unusually short distance (1.84 (2) Å) compared to W2 (2.03 (1) Å), which may indicate a hydroxide
(OH-) ligand. The second T2Cu(II) active site conformation also reveals a completely unique
architecture, extending from the observations of the previous structure which revealed the presence of
lower distorted proximal conformation of Asp (Asp98).
CAT
In the current atomic resolution XFEL structure, this lower distorted conformation of Asp (65%
CAT
occupancy), is rotated 38° around the Oδ2 atom, facing much lower towards the Cu atom and
consequently allowing the Oδ2 atom to be positioned only 2.20 (2) Å away from the T2Cu(II), a distance
indicating direct interaction with T2Cu(II) (Fig. 5d). It occupies the site with W2 when W1 is not
present – in 2 monomers/homotrimer. When this unique T2Cu coordination of Asp is not observed,
CAT
the classical proximal position is retained together with W1 (35 %) and W2 at the T2Cu(II) site. W3 is
also observed within the active site once more, linking with the fully occupied W2 ligand and proximal
Asp conformation. His is rotated towards Thr280 in this structure, ensuring suitable distances for
CAT CAT
hydrogen bonding to both Thr280 (2.77 Å) and Glu279 (2.67 Å)

At the T1Cu(II) redox centre, the tetracoordinated site is consistent with the previous XFEL structure,
with coordination distances (Supplementary table 2 and Supplementary Fig. 4e) in agreement with a
fully-oxidised redox state as also confirmed by the single crystEal optical spectrum (Fig. 3c). Slight
shortening of the Met-T1Cu(II) distance and elongation of the Cys-T1Cu(II) is observed, common for
the green subset of T1Cu proteins. Comparison with th e oxidised T1Cu(II) of BrNiR, reveals a slight
shift in geometry as a result (Supplementary Fig. 4i).

Nitrite-bound oxidised AcNiR (pH 4.8)L at 0.95 Å resolution. The sub-atomic resolution XFEL
structure of nitrite-bound oxidised AcNiR (pH 4.8) was determined to 0.95 Å resolution (Supplementary
Table 1,2). The single crystal optTical spectroscopy prior to collection of XFEL data confirmed that
enzyme was in the oxidised Cu(II) state (Fig. 3c). When compared to the previously collected lower
resolution cryogenic XFEL structure (1.5 Å resolution)15, several previously unseen features are
observed. In the 0.95 Å resolution structure (Fig. 6a,b) two binding modes of the nitrite were observed
at the T2Cu(II) active site with compatible Asp conformation (proximal/gatekeeper) (Fig. 6b), with
CAT
these evident in the Fo-Fc OMIT maps (Supplementary Fig. 1f). The higher resolution structure
combined with SHELXL refinement, shows two variations of the “top-hat” binding mode, as opposed to
a “top-hat” and “side-on” binding mode previously modelled in the lower resolution SF-ROX structure.
The major occupancy “top-hat” nitrite conformation (65 % occupancy) (Fig. 6c) has almost equal N-O
1
bond lengths (~1.28 Å; O1-N [1.29 (3)]/O2-N [1.28 (3)] and an angle of 111° (2)) and exhibits bidentate
coordination to the T2Cu(II) via the two oxygen atoms (O1: 1.88 (2) Å, N: 2.42 (3) Å, O2: 2.14 (2) Å)
(Supplementary Table 3 and Supplementary Fig. 2f). This conformation correlates with the proximal
position of Asp (68 % occupancy) due to active site steric restraints but the O2 atom of the molecule
CAT
is more suitably positioned to the Nε2 atom of His (3.06 Å) for hydrogen bond formation compared
CAT
to Asp . This active site conformation occupies ~ 2 monomers/homotrimer. Other partial waters are
CAT
also observed in this active site conformation (W and W4) with these linking to the nitritemolecule.
p
The minor occupancy “top-hat” nitrite conformation (35 % occupancy) (Fig. 6d), ~1
2
monomer/homotrimer, also has almost equal N-O bond lengths (~1.27 Å; O1-N [1.28 (3)]/O2-N [1.27
(3)] and an angle of 120° (2)) and again exhibits bidentate coordination to the T2Cu(II) via its two
oxygen atoms (O1: 2.11 (2) Å, N: 2.40 (2) Å, O2: 2.04 (1) Å) (Supplementary Table 3 and
Supplementary Fig. 2f). This conformation now correlates with the gatekeeper position of Asp (32 %
CAT
occupancy) and a new partial occupancy water (W ; 33 %) which takes the place of the second carboxyl
G
oxygen (Oδ2) atom of Asp , which is at a more realistic distance foSr hydrogen bond formation with
CAT
the O2 of nitrite than Asp (2.71 Å compared to 2.01 Å). The observation of two conformations of
CAT
nitrite, both with a “top-hat” binding mode, alongside two cRonformations of Asp
CAT
, provides
persuasive evidence that the “top-hat” binding mode is thPe initial binding mode of nitrite before the
beginning of catalysis – in contrast with our previoNus suggestion of a “side-on” being the initial
coordination mode16. His is similarly rotated t I owards Thr280 like the as-isolated AcNiR structure,
CAT
with suitable distances for hydrogen bonding to both Thr280 (2.77 Å) and Glu279 (2.70 Å)

At the T1Cu(II) redox centre, distanIces for the tetracoordinated site are almost identical to the as-
isolated oxidised state (SupplRementary table 2 and Supplementary Fig. 4e). Comparison with the
equivalent state in the bluiAsh-green BrNiR, revealed a similar shift in geometry as the BrNiR and AcNiR
as-isolated oxidised species and confirmed that the slight disorder of the Met ligand observed in the
tetracoordinated T1Cu(II) site in BrNiR is representative of a mixture of blue and green Met positions
(Supplementary Fig. 4j)

Single crystals spectroscopy of copper-nitrite reductase species
Single crystal spectroscopy at 100 K on the same crystalline sample was also performed prior to SF-
ROX measurements at SACLA to confirm the valence state of the T1Cu redox centre. Fig. 3 shows the
optical spectra from single crystals of bluish-green CuNiRs from two Bradyrhizobium species
(Bradyrhizobium sp. ORS 375 referred to as Br2DNiR (Fig. 3a) or Bradyrhizobium japonicum referred to
as BrJNiR (Fig. 3b)) at pH 5.5 and pH 7.3 and prototypic green CuNiR, AcNiR at pH 4.8 (Fig. 3c). For
the as-isolated crystals of each CuNiR, after soaking with same buffer conditions and cryoprotectants as
that used for the SF-ROX measurements, confirmed for all cases the T1Cu redox centre were in their
fully oxidised Cu(II) states. For comparison, spectra for solution Br2DNiR at pH 6.5 are also included
(Fig. 3d). In all cases, optical spectra of the oxidised enzyme shows the bands that have been reported
for a variety of CuNiRs that give rise to their colours, blue, green and bluish-green11. In comparison to
the solution spectra at room temperature (Fig. 3d), single crystal spectra taken at ~100 K are much
sharper where some of the weaker broad bands become well resolved. For example, the broad feature
between 650-800 nm that have been assigned to d-d transition, becomes well resolved bands at ~680 nm
and ~770 nm. Likewise, the shoulder at ~380 nm, assigned to a S(Met) → Cu ligand-to-metal charge
transfer (LMCT), resolves into a clear peak. Similar sharpening has been seen for CuNiR from
Rhodobacter sphaeroides, (RsNiR) for solution samples when cooled to 7 K10. Relative intensities of
~570 nm, assigned to S(Cys) → Cu  CT transition and ~460 nm, assigned to S(Cys) → Cu  CT band,
gives rise to the colour with AcNiR being green and BradyrhizobiSum CuNiRs (Br2DNiR and BrJNiR)
being bluish-green. Addition of nitrite to the crystals of oxidised enzymes (with same soaking procedure
as XFEL data collection) shows a band associated with a LMCT band of nitrite at ~350 nm. It is clearly
resolved in the single crystal spectra of both Bradyrhiz obium species but for AcNiR it overlaps with the
380 nm band. Upon chemical reduction of the oxidised enzyme crystals, all the optical bands for these
CuNiRs are bleached, consistent with T1Cu being in Cu(I) oxidation state. Addition of nitrite to these
reduced crystals results in small recoveryL of the optical bands irrespective of the duration of the soak.
We note that the length of the long soak was significantly more than the soaking time used for soaking
nitrite to the oxidised crystals that yielded full occupancy of nitrite at the catalytic T2Cu site. The low
recovery of T1Cu optical spectrum (~20 %) indicates that binding of nitrite to the reduced T2Cu(I) site
is limited but not prevented, consistent with earlier observations on CuNiR from Alcaligenese
xylosoxidans (AxNiR)33, providing clear evidence for the binding-before-reduction pathway as opposed
to the reduction-before-binding pathway34, which also agrees with the structural observations of the
mixed T2Cu(I) sites that are observed here in the atomic resolution XFEL structures of the dithionite-
reduced BrNiR, with one maintaining a water ligand capable of binding nitrite via displacement of the
water and the consequent reoxidation of the T1Cu resulting from turnover, and the other representing
the inactive dead-end (T2Cu(His ) state.
3

Protonation states for the catalytic aspartic and histidine residues from SHELXL refined accurate
atomic resolution single shot XFEL structures
With the cardinal combination of atomic resolution damage-free structures obtained on crystals whose
oxidation state have been validated by cryogenic single crystal spectroscopy prior to single shot XFEL
pulses (< 10 fs), together with SHELXL refinement, a unique opportunity is provided to assess the
protonation states of the two catalytic residues in these CuNiRs (Asp and His ) during some of the
CAT CAT
key catalytic states. This is enabled by a final step of unrestrained refinement to remove the influence of
crystallographic dictionary restraints and for estimation of standard uncertainties (esds) of bond lengths
and angles. We originally applied a similar method for atomic resolution synchrotron structures of the
BrNiR from Bradyrhizobium sp. ORS 375, using predication methods previously suggested, where in
short: data from the Cambridge Structural Database (CSD)44 has suggested that for aspartate residues
the bond lengths between Cγ─Oδ1 and Cγ─Oδ2 are expected to be equal to ~ 1.256 Å when non-
protonated and when protonated one bond will increase to ~ 1.310 Å and the other will become a double
bond with a distance of ~ 1.210 Å45. For histidine residues the bond angles are used to assign its
protonation state and the differences between protonated and nEon-protonated states relatively small, so
greater caution is required. An analysis of imidazole moieties in histidine residues from atomic
resolution structures in the CSD and the Protein Data B ank (PDB)46 has suggested that histidine is fully
protonated if the bond angles of Cε1-Nε2-Cδ2 and Cγ -Nδ1-Cε1 are greater than 108.9 ˚ 47. In our current
analysis, we estimate that His is protonated if the bond angles of Cε1-Nε2-Cδ2 and Cγ -Nδ1-Cε1 are
CAT
greater than 107˚. In contrast to the electrLon density maps obtained in the atomic resolution structures
using storage ring X-rays 48, no hydrogen atoms were observed in neither 2F -F or F -F maps,
o c o c
presumably because of the pulseTd nature of XFEL (pulse to pulse variations) and large number of
crystals each with multiple spots used for data collection. Despite the lack of guidance from the
experimental density maps, the analysis of the Cε1-Nε2-Cδ2 and Cγ -Nδ1-Cε1 angles of His
CAT
(Supplementary Table 4) allowed us to conclude that this residue is likely to be fully protonated in all
structures. There are differences in Asp protonation depending on the catalytic state of the protein. In
CAT
the pH 5.5 oxidised BrNiR structure, the Oδ2-Cγ 1.27 (2) Å bond length is slightly longer than Oδ1-Cγ:
1.25 (2) Å, which may be due to light protonation on Oδ2, making it almost neutral. In the dithionite-
reduced BrNiR structure (pH 5.5) the Oδ2-Cγ: 1.29 (2) Å is more elongated, which indicates the potential
full protonation on Oδ2 and thus not negatively charged. For the high pH oxidised (pH 7.3) and nitrite
bound (pH 5.5) BrNiR structures, Asp appears to be non-protonated (both bond lengths are equal), or
CAT
negatively charged. In AcNiR, for both the nitrite-bound (pH 4.8) and the oxidised (pH 4.8) forms,
Asp in its proximal conformation appears to be non-protonated and negatively changed. However, in
CAT
the lower distorted Asp conformation in the oxidised structure (pH 4.8), there is large asymmetry in
CAT
the bond lengths of Asp (Oδ1-Cγ: 1.24 (3) Å, Oδ2-Cγ: 1.34 (4) Å), which would indicate Oδ2
CAT
protonation, with Oδ2 creating the direct bond with T2Cu at a distance of 2.2 (1) Å. Supplementary
Tables 4 and 5 summarises the bond lengths, angles and protonation states for these catalytic residues.

Methods
Expression and purification of recombinant BrNiR and AcNiR
Expression and purification of recombinant BrNiR species into Escherichia coli (E. coli) were
performed according to established procedures for BrJNiR25,32 and Br2DNiR16,28. Likewise, expression
and purification of recombinant AcNiR into E. coli was performed using established procedures15.
Samples were buffer exchanged into a crystallisation buffer consisting of 10 mM HEPES pH 6.5
(BrNiR) or 10 mM MES pH 6.5 (AcNiR) prior to crystallisation.

Crystallisation of BrNiR and AcNiR and preparation for XFEL
Crystals of BrNiR types and AcNiR were grown using the vRapor diffusion hanging drop method at room
temperature with 400 µL reservoir solution consisting of 1.8 M ammonium sulphate, 50 mM HEPES
(pH 5.0) for BrNiR (Br2DNiR); 1.8 M ammonium sNulphate, 100 mM Tris (pH 7.3) for BrNiR (BrJNiR);
and 1.2 M ammonium sulphate, 50 mM citrate (pH 4.8) for AcNiR, respectively. For BrNiR, ~ 25
mg/mL protein solution was mixed 2:1 with reservoir solution. Crystals grew after a few days - for
BrNiR (Br2DNiR), cubic-shaped crystCals grew in space group P2 3 and for BrNiR (BrJNiR), crystals
1
grew in space group P6 . For AcNiR, ~ 20 mg/mL protein solution was mixed 1:2 ratio with reservoir
3 T
solution and pyramidal-shapeRd crystals grew after a few days in P2 3 space group. BrNiR crystals were
1
transferred to a storage solution of 2.5 M ammonium sulphate, 50 mM HEPES (pH 5.5) and AcNiR
crystals were transferred to a storage solution of 2.5 M ammonium sulphate, 50 mM citrate (pH 4.8)
before preparation for XFEL experiments.

For the as-isolated (resting state) structure of BrNiR (Br2DNiR) at pH 5.5 – 42 individual crystals
ranging from 400 µm3 – 700 µm3 were prepared for XFEL collection with crystals soaked in a
cryoprotectant solution consisting of 3.3 M ammonium sulphate, 50 mM HEPES (pH 5.5) and 20.3 %
sucrose prior to cryocooling by plunging into liquid nitrogen. For the high pH as-isolated (resting state)
structure of BrNiR (BrJNiR) at pH 7.3 and nitrite-bound structure of BrNiR (BrJNiR) at pH 5.5 - 64
individual crystals ranging from 500 µm3 – 700 µm3 were prepared for XFEL collection, respectively.
For the high pH BrNiR (BrJNiR) resting state, crystals were soaked in a cryoprotectant solution
consisting of 2.8 M ammonium sulphate, 50 mM Tris (pH 7.3) and 17.5 % sucrose for roughly two
minutes to ensure successful pH increase prior to cryocooling by plunging into liquid nitrogen. For the
nitrite-bound BrNiR (BrJNiR) state, crystals were soaked in a nitrite soaking solution consisting of 2.8
M ammonium sulphate, 50 mM HEPES (pH 5.5) and 200 mM sodium nitrite before being transferred to
a cryoprotectant solution consisting of 2.8 M ammonium sulphate, 50 mM HEPES (pH 5.5), 200 mM
sodium nitrite and 17.5 % sucrose prior to cryocooling by plunging into liquid nitrogen. For the
chemically-reduced structure of BrNiR (Br2DNiR) at pH 5.5 – 64 individual crystals ranging from 400
µm3 – 500 µm3 were prepared for XFEL collection with crystals soaked in a cryoprotectant solution
consisting of 2.8 M ammonium sulphate, 50 mM HEPES (pH 5.5), 100 mM dithionite and 17.5 %
sucrose prior to cryocooling by plunging into liquid nitrogen. Crystals changed colour from bluish-green
to colourless during soaking indicating successful reduction of the T1Cu from Cu2+ to Cu+. 1M stock
solution of HEPES was titrated with HCl to pH 5.5 before mixing with other chemicals for preparation
of crystallisation solutions and soaking solutions.

For the as-isolated (resting state) structure of AcNiR at pH 4.8 and nitrite-bound structure of AcNiR at
pH 5.0 - 48 individual crystals ranging from 400 µm3 – 500 µm3 were prepared for XFEL collection,
respectively. For the resting state, crystals were soaked in a cryoprotectant solution consisting of 2.8 M
ammonium sulphate, 50 mM citrate (pH 4.8) an d 17.5 % sucrose prior to cryocooling by plunging into
liquid nitrogen. For the nitrite-bound statLe, crystals were soaked in a nitrite soaking solution consisting
of 2.5 M ammonium sulphate, 50 mM citrate (pH 5.0) and 200 mM sodium nitrite before being
transferred to a cryoprotectant soTlution consisting of 2.5 M ammonium sulphate, 50 mM citrate (pH
5.0), 200 mM sodium nitrite and 17.5 % sucrose prior to cryocooling by plunging into liquid nitrogen.

Automatic data collection pipeline of XFEL data at SACLA
All data were collected using higher photon energy (13 keV) at SACLA to push the achievable
resolution. Collection was performed on BL2/EH3 at SACLA at 100 K using the serial femtosecond
rotational crystallography (SF-ROX) technique49. The diffraction images were collected on a MX300-
HS detector (Rayonix) with a camera length of 90 mm and a pixel size of 78.2 μm. As-isolated (resting
state) data of BrNiR (Br2DNiR) at pH 5.5 was collected before the implementation of the automatic data
collection pipeline of XFEL data at SACLA. Here, data collection of 41/42 crystals prepared was
performed using the SF-ROX technique 49. The following parameters of XFEL pules were used: pulse
duration (<10 fs), pulse energy (~ 232 µJ), flux (~ 4 x 1012 photons), beam size at sample position (3.91
μm (H) by 4.14 μm (V)). The crystals were rotated 0.15° and translated 50 μm between each shot and a
silicon attenuator with thickness of 0.22 mm was used.

For all other data sets, the automatic data collection pipeline was used. Here, briefly: four unipucks are
loaded into the sample exchange robot (SPACE-II)55 [each individual unipucks must have the same loop
sizes], the unipuck information is updated on the beamline scheduling software (BSS)56, for each loop
size a set number of points are determined, together with the rotation range (e.g. for a 0.5 mm loop: 12
(vertical) x 15 (horizontal) with a rotation range of 1.5° was used (Number of horizontal points x 0.1°
oscillation)). The camera distance is selected, and an auto-schedule is generated which then
automatically performs sample centring and collection for each unipuck using the SF-ROX approach.
The automatic collection of one unipuck (i.e. 16 crystals) took ~ 1.5 hours. The following parameters of
XFEL pulses were used for all: photon energy (13 keV), pulse duration (<10 fs), pulse energy (~ 205
µJ), flux (~ 4 x 1012 photons), beam size at sample position (3.49 μm (H) by 4.14 μm (V)). The crystals
were rotated 0.1° and translated 50 μm between each shot. All Ediffraction images were collected on a
MX300-HS detector (Rayonix) with a camera length of 89.2 mm and a pixel size of 78.2 μm and an
aluminium attenuator with a thickness of 0.2 mm was u sed (corresponding to 54 % transmission at 13
keV) throughout.

Data processing and unrestrained refinLement
On-the-fly hit finding was performed using the SACLA processing pipeline, which incorporates
Cheetah57. Data processing was pTerformed using CrystFEL58 version 0.9.1 with spot finding and
indexing performed using the peakfinder8 algorithm and XGandalf 59, DirAx 60 and Mosflm61,
respectively in indexamajig, with the following parameters (--threshold=10, --min-pix-count=2, --min-
snr=15, --local-bg-radius=3). The inner, middle, and outer integration radii were set to 6, 7, and 8 pixels,
respectively. Indexing ambiguity was resolved using ambigator using the operator k, h, -l for both P2 3
1
(m-3) crystals and P6 (6/m) crystals. Bragg intensities were merged using the Monte Carlo method
3
with process_hkl, with the high-resolution limit (d−1) extended by 2.3 nm−1, 3.0 nm−1, 3.5 nm−1, 4.0
nm−1 using the --push-res option for low-pH BrNiR, high-pH BrNiR and low-pH AcNiR, nitrite-bound
BrNiR and chemically-reduced BrNiR, and nitrite-bound AcNiR, respectively.

For refinement of anisotropic displacement parameters (ADPs), SHELXL30 was used. Reflection files in
MTZ format and template starting models in PDB format (PDB codes: 6ZAS
[http://doi.org/10.2210/pdb6ZAS/pdb] (BrNiR (Br2DNiR), 8R8S [http://doi.org/10.2210/pdb8R8S/pdb]
(BrNiR (BrJNiR) and 6GSQ [http://doi.org/10.2210/pdb6GSQ/pdb] (AcNiR)) were first converted to
HKLF4 and INS SHELX formats using mtz2hkl62 and pdb2ins63 programmes, respectively. Several
rounds of restrained refinement were performed with isotropic B-factors, with incremental increases in
resolution up to the resolution limit. Refinement was interleaved with manual model building using
Coot64 - with ligands, additional conformations of side chains and waters added after each stage of
refinement. For all structures, occupancies of single conformation residues and ligands were set to one
and adjusted manually based on the electron density. The occupancies of residues with double
conformations were set to 50 % and then refined in SHELXL, using the FVAR command. For the
BrNiR dithionite-reduced structure, both T1Cu and T2Cu positions and the single solvent water were
also automatically refined using the FVAR command in SHELXL. For AcNiR structures, only the
occupancies of Asp conformations were automatically refined in SHELXL using the FVAR
CAT
command. Anisotropic B-factors were then refined, together with the addition of hydrogen atoms and at
the final stage of the refinement, one cycle of unrestrained refinEement (BLOC 1 style) was implemented
to estimate the standard uncertainties (e.s.d.s) of coordinates and derived parameters (bond lengths and
angles). Ellipsoidal models for all structures showing A DPs with 50 % probability were also produced
using the ShelXle GUI65, with these shown in Supplementary Fig. 5. Data collection and refinement
statistics are shown in Supplementary Table 2.

Single crystal and solution UV-vis absorption spectroscopy
Single crystal UV-vis absorptionT spectroscopy measurements were obtained off-line at SPring-8 at 100
K using a fiber-optic microspectrophotometer with linear charge-coupled device array detector (Ocean
Optics, SD2000). Single crystals of Br2DNiR, BrJNiR and AcNiR with dimensions ~400 µm3 were used
for measurements. Crystals were transferred to a storage solution as described above prior to crystal
soaking. For BrNiR crystals, these were soaked in 2.8 M ammonium sulphate, 50 mM HEPES (pH 5.5)
[+ 200 mM sodium nitrite or 100 mM sodium dithionite] before being soaked in same solutions with
17.5 % sucrose for use as a cryoprotectant. and for high pH measurements, these were soaked similarly
but with a buffer of 50 mM Tris pH 7.3 instead and for longer incubation times to allow for pH increase.
For AcNiR crystals, these were soaked in 2.8 M ammonium sulphate, 50 mM citrate (pH 4.8) [+ 200
mM sodium nitrite or 100 mM sodium dithionite] before being soaked in same solutions with 17.5 %
sucrose for use as a cryoprotectant. For the experiment involving nitrite soaking of reduced crystals,
these were performed on the chemically-reduced cryoprotected colourless samples, which were in
T1Cu+ reduced state. Following reduction, crystals were transferred to a cryoprotectant solution
containing 200 mM sodium nitrite. For short soaking, crystals were soaked ~ 20 seconds before being
cryocooled by plunging into liquid nitrogen – which would cryotrap the state. For long soaking, crystals
were soaked ~ 3 minutes before being cryocooled by plunging into liquid nitrogen – which would
cryotrap the state. Solution UV-vis absorption spectroscopy measurements were obtained using a Cary
3500 Compact UV-Vis Spectrophotometer (Agilent) with a quartz cuvette of 10 mm pathlength. A final
protein concentration and buffer of 26 µM in 50 mM HEPES pH 6.5 was used for all measurements and
a reference containing just 50 mM HEPES pH 6.5 buffer was taken prior to measurements for
subsequent background subtraction. For nitrite addition, a final concentration of 10 mM sodium nitrite
(NaNO ) was added to the protein solution. Likewise, to reduce the enzyme, a final concentration of 1
2
mM dithionite was added. 10 mM NaNO was added to the reduced form of the enzyme for the
2
additional experiment with collection started immediately after this.

All spectra were processed using the icOS-toolbox app66. The aEverage absorbance between 500 and 510
nm was subtracted from every spectrum for baseline correction. Corrected spectra were plotted using
the Igor Pro software package (Wavemetrics).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by S. Samar Hasnain and colleagues is one of the first comprehensive studies of copper nitrite reductases,\nexamining different oxidation states, and substrate binding at different pH values using X-ray energy. The atomic-resolution\nstructures presented here provide valuable insights into the substrate-binding mode and the coordination of the two copper\nsites, which are of great importance to researchers working with nitrite reductases and other copper metalloproteins. The\nability to attain information on resting and substrate-bound states also makes this study relevant to those working with\nenzymes.\nThe manuscript presents a substantial amount of new data and is generally well-written and clearly presented. The number\nand quality of figures are appropriate for the scope of the study.\nHowever, there are several issues that require clarification or correction before the manuscript can be considered for\npublication.\nNote: As the manuscript lacks page numbers, all comments below will be referenced to line numbers.\n1. The abstract should be revised to present the main conclusions obtain in this study.\n2. Line 50: Role in Nitrification\nThe authors state that the enzyme is a key enzyme in the nitrification pathway. To the best of my knowledge, this enzyme is\nprimarily involved in the denitrification pathway. I would appreciate a clarification and appropriate referencing on how the\nenzyme fits into nitrification, or a correction if this was a misstatement.\nReference 3 is not appropriate to support this sentence. Please revise your references throughout the manuscript.\n3. Lines 56 to 70: Missing references\nThis section includes several assertions and background details that should be supported by citations. Please provide\nappropriate references to substantiate the statements made here.\n4. Classes of Nitrite Reductases – Clarify Earlier\nThe different classes of nitrite reductases used in this study are only introduced later in the manuscript (Line 395). For clarity\nand better flow, I strongly suggest that the authors introduce this classification earlier, ideally in the Introduction or at the\nbeginning of the Results section. This would provide readers with the necessary context to interpret the visible spectra in\nFigure 3, that is first referenced in Line 221.\nIt would also be important to mention why these classes were chosen.\n5. Methods: Missing information\nThe Methods section lacks essential details about the protein samples used for crystallization. In particular, the authors\nshould provide more information on the metal content of the proteins, including the number of copper atoms bound per\npolypeptide chain. Additionally, data on protein purity, such as absorbance ratios, and the specific activity of the enzyme\nshould be reported to confirm that the crystallized protein was functionally competent. The occupancy of the copper site\nobtained from structural refinement should also be included in the manuscript text, as this information is important to support\nthe structural interpretation of the active site.\n6. Line 155: Bound hydroxide at low pH\nThe manuscript reports a bound hydroxide ion at pH 4.8, yet it does not offer an explanation for the presence of OH⁻ at such\nan acidic pH. This observation is unusual and warrants further discussion, particularly with regard to the possible\ninvolvement of a nearby residue that might change the local pKa, or to stabilization through metal coordination. Can the\nauthors mention the occupancy to identify it was an oxygen atom.\n7. Line 188: Coordination of T1Cu BrNiR\nIn the discussion of the T1Cu center in BrNiR, the authors mention that Met145 adopts two positions in the structure solved\nat pH 7.3 and in the nitrite-bound form. However, no mechanistic or structural rationale is provided for this dual conformation.\nAlthough they cite literature (line 189-190) linking the axial methionine's position to the reduction potential of the T1Cu sites,\nthey do not directly relate this to the properties of their own proteins.\nFurthermore, such difference in geometry of the center should be observed in its spectroscopic\nfeatures, such as visible absorption spectra, visible CD or EPR spectra. The visible spectra obtained from the crystals should be better analyzed to\ncorroborate this observation. Moreover, the authors should mention whether this effect has been observed in the structures\nof other T1Cu proteins.\nCan the authors explain whether such conformations cannot be attributed to low copper occupancy?\n8. Line 425-427: Catalytic Mechanism\nThe reviewer can agree that, in the mechanism of these enzymes, nitrite binds before the electron is delivered, so that it\nbinds to the oxidized T2Cu center (with or without T1Cu center reduced). However, the reviewer disagrees with the authors'\nexplanation and interpretation of the spectroscopic data. The authors claim that the low recovery of the T1Cu absorption\nfeatures means that nitrite has to bind before T2Cu is reduced. However, the recovery of the T1Cu absorption features\ndepends on the relative amounts of the reducing agent and nitrite, as well as on the catalytic rate constant. The time at which\nthe spectra were collected after the addition of nitrite is also a factor.\nTherefore, to support their hypothesis of “nitrite binding-before-reduction”, the authors could perform the reaction in reverse\norder (add nitrite to oxidized NiR and then dithionite) and collect spectra at the same time points as before.\n9. pKa of the bell-shaped curve\nThe authors study the structure of what they designate as “high” and “low” pH. Most low pH falls below or are the optimum\npH of these enzymes, with high pH above it. It is important to note the pKas of the pH dependence of the catalytic activity of\neach enzyme is not reported and it would help the interpretation of the data. Additionally, the authors fail to establish a clear\ncorrelation between the protonation state of AspCAT and HisCAT during catalysis. Form their data AspCAT only becomes\ndeprotonated after nitrite binding and at high pH, whereas HisCAT is always protonated as required for activity. Their\nstructures would correspond to the optimum pH structures, but further data on proton transfer is not made.\n10. Low pH structures\nAt this pH, is the protein damaged? Is the stability of these enzymes high at such a low pH?\nLine 497: Are these low pH values the optimum pH of the enzymes under study? Please provide a reference.\n11. Use of HEPES | MES\nThe authors mention several times in the Methods section to have used HEPES pH 5.0 or 5.5, this is a typo for sure, as the\npKa of HEPES is around 7.5 and at such low pH it does not have buffering capacity. So, in which conditions HEPES was\nused?\nFigures and Figure Legends\nIn the Figure legends add for all cases the indication of which pH the protein was crystallized (including Figure 5).\n1. Line 200: Legend of Figure 2\nMention in the Figure legend the meaning of BW. As far as I can see GK in not in this figure. The figure legends need to be\nrevised.\n2. Line 299: Legend of Figure 4\nPanel (b) seems to still show the two conformations in cyan and blue. Was this done on purpose?\n3. Line 390: Legend of Figure 6\nThe Figure legend mentions LD, which is not in the figure. Please indicate the meaning of water WG and GK.\nOther corrections\n1. Page 46, The c as in cytochrome c should be in italic (please check the text for other occurrences).\n2. Page 53, cd1, 1 is subscript and not in italic\n3. Line 353, indicate at which pH the protein was crystallized.\n4. Line 401: japonicum without capital letter\n5. Line 438-440: sentence needs to be revised\n6. Line 453-456: “… allowed us to conclude that these residues …” residue or residues, seems to be related only to HisCAT.\n7. Line 485 (and other below): penta-coordinate substitute for penta-coordinated\n8. Line 485: revise “tris-his coordination and two T2Cu ligands” – not usual designation and missing words?\n9. Line 486: and a hydroxide\n10. Line 558 (and other): microL or mg/mL (correct the Liter as capital letter)\n11. Line 675: concentration and buffer\nThe designation microm, is a typo for microM? The buffer used was really HEPES pH 6.5? It would be on the limit of the\nbuffering capacity. The spectra were collected after nitrite addition to the reduced protein at which time points? This should\nbe mentioned in the Figure legend. The same should be mentioned for the oxidized form incubated with nitrite. Please check\nmy comment above.\n12. Line 686: to be removed?\n13. References\nThe reference style needs to be revised as it is not homogenous throughout the document: different fonts used, different\nformats, missing DOI for most, name of the journals not uniform, bold year or volume, date sometimes in parenthesis, titles\nwith capitalized words in some cases and in other not, microorganism’s name should always be in italic.", "reviewer_2": "This manuscript presents, to the best of our knowledge, the first atomic resolution XFEL structures of copper nitrite\nreductases (CuNiRs) in multiple functional relevant states, as oxidized, reduced, and nitrite-bound forms. By exploiting high-\nenergy (13 keV) femtosecond X-ray pulses at SACLA and refining with SHELXL, the authors achieve structures free from\nradiation-induced chemistry, overcoming a major limitation of synchrotron studies where photoreduction distorts the copper\nsites. The work integrates crystallography with single-crystal spectroscopy to confirm the redox state of the type 1 copper site\nand supports new mechanistic insights into nitrite binding modes, water/hydroxide coordination, and the protonation states\nof key catalytic residues, such as Asp and His . These findings may have broad implications for understanding\nCAT CAT\nproton-coupled electron transfer, substrate gating and binding and reactivity in CuNiRs. Moreover this manuscript represents\nthe first application of SHELX, a suite of crystallographic software, to XFEL atomic resolutions structures. The manuscript is\nwell-written and the methods described in the main text, and the details in the supporting information, are enough detailed to\nallow reproduction. Overall, the work is exciting and deserve to be published in Nature Communications journal after\nclarification of the following points:\n1) Figure 4 d, e, f are almost certainly type 1 not type 2 Cu sites as they contain cys and met residues. I find it hard to believe\nthat if the coordination number of the copper changes (losing a water) and the copper shifts positions that the imidazoles of\nthe ligands also do not rotate. Figure 4b implies they don’t. If the authors want to really compare these structures they need\nto show conformations of all atoms at the active site. Figure 4d seems to account for these differences.\n2) Page 18, they are inferring protonation states from bond lengths and angles not determining protonation states. The latter\ncase, actual seeing the protons would require a neutron structure.\n3) We get the point that these are “the first” structures. The authors should only state this once and then focus on what their\nresults mean. The reader feels beaten over the head with this emphasis on novelty when this statement is constantly\nrepeated in abstract, introduction, results and discussion.\n4) The abbreviation for the bridging water is sometimes inconsistent. On page 7 it is written as W (with Br as subscript),\nBr\nwhile in Figures 2a, 2b, and 2c it is labelled as BW. The notation should be standardized along the text and figures.\n5) As inferred by the authors in the Figure 2 captions, the loop connecting the His ligands of the T1Cu and T2Cu sites is\nproposed to act as a “sensor” in nitrite binding. Given the atomic resolution of these structures, it would be nice to know\nwhether the authors compared the conformation of this sensor loop across the different functional states (oxidized, reduced,\nnitrite-bound). For example, a analysis of the RMSD calculated on the Calfa atoms of this loop could highlight small relevant\nrearrangements. Including such a comparison may strengthen the argument for its role in substrate binding.\n6) How the authors determined the occupancies of residues around the T1Cu and T2Cu centers? It is not entirely clear from\nthe methods. Were the occupancies adjusted manually based on the electron density, or were restraints applied to refine?\nGiven the importance of correctly modeling partial occupancies near the active site, a more detailed explanation in the\nMethods would be very helpful. The inclusion of specific refinement criteria to justify alternate conformations and their\npercentage would increase the strength of the results. In addition, including Fo-Fc omit maps in the supporting information\ncould further support the presence of alternate conformations or partial occupancies.\n7) In the BrCuNiR structure at pH 5.5 (Figure 2a), His250 appears to be involved in a different hydrogen-bonding network\ncompared to the pH 7.3 structure (Figure 2b). Could the authors clarify whether they observe any difference in the orientation\nof His250 between these two conditions? Additionally, what is the role of the bridging water at pH 7.3? Does it form new\nhydrogen bonds respect the structure at pH 5.5? These points should be addressed, as subtle structural differences may\nprovide a structural rationale for the lower catalytic activity observed at neutral pH.\n8) The authors state that all structures were collected at 100 K. However, at cryogenic temperatures protein flexibility,\nhydrogen-bonding interactions etc. can differ significantly from those occurring in solution under physiological conditions. A\nshort discussion of how cooling may influence the observed hydrogen-bond network, nitrite binding, and mechanistic\ninterpretations would need to be added.\n9) The primary concern with the paper is not in the results, which are highly significant, but in the lack of broader analysis in\nthe discussion. Now that we have all these novel structures, what do they tell us about the catalytic mechanism of the\nsystem? We are given exhaustive detail on structures of both type 1 and 2 Cu centers, on the substrate bound forms and on\nvarious pH conditions. But in my opinion, none of this data is brought together to assess catalytic mechanism for nitrite\nreduction that have been proposed. For example, is proton gating critical and how is it achieved both by changing the\ncatalytic site and also the electron transfer site? Are structural changes observed consistent with known reduction potential\nchanges in these systems based on substrate binding and protonation? How is the nitrite conformation important for\nproposed mechanisms (they hint at this in Fig 2 results showing gatekeeper conformations but never discuss how this\ninfluences the type 1 center structure and hence potential)? I could go on… The authors have really addressed excellently\ncritical structural issues with this work; however, they have dropped the ball when tying the importance of their discoveries to\nthe known catalytic system. This discussion is what is required to elevate this article to the level of Nat Comm, otherwise, the\nwork should be in a more specialized, but still high impact crystallographic journal. With the proper discussion, I would fully\nsupport Nat Comm publication.\nMinor issues:\n- Typos along the text like “SHLEXL” in the abstract\n- Mixing british and american English spellings (e.g., oxidised vs oxidized, modelled vs modeled). Please standardize to one\nstyle, in line with the journal guidelines.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "The authors report several atomic resolution crystal structures of copper nitrite reductase (CuNiR) from Bradyrhizobium and\nAchromobacter species obtained using an X-ray free electron laser (XFEL), providing detailed and radiation damage-free\nviews of the two metal centers in these redox proteins. The authors used an approach where they rotate and serially expose\nmultiple regions of larger crystals to the XFEL beam at cryogenic temperature (SF-ROX), which permits using many fewer\ncrystals to obtain a complete dataset. SF-ROX has the clearly demonstrated potential to generate data whose resolution\nexceeds that typically obtained using more conventional serial delivery of randomly orientated microcrystals typically used at\nXFELs. The authors have previously published pioneering work on this method and applied it to CuNiR enzymes (PMID:\n29354268, 31316819). Therefore, this is an established method and the principal advance being reported in this manuscript\nis obtaining markedly improved resolution data for CuNiRs from multiple species. This improved resolution allows them to\nrefine the models in SHELXL, which can produce estimated standard uncertainties (ESUs) on model parameters. Overall,\nthis is an impressive example of crystallography at XFEL sources being used to answer questions that are not easily\naccessible using other X-ray sources owing to radiation damage.\nDespite its technical strengths, the central findings of the study are difficult to summarize, as they cover the details of the\nchanges in the metal and coordinating residue geometries as a function of species, pH, and redox state. In support of this\nimpression, the Abstract provides unusually little sense of what was learned in the work. While the results are discussed in\nexhaustive detail, it is unclear (at least to this reviewer) what the core general message of the manuscript is. For example, I\ncannot confidently determine what a reader (particularly one who is not actively working on the structural biology of CuNiRs)\nmight take from this study. This is at least partially the result of a difficult writing style, focused on providing lists of\ninteratomic distances in multiple proteins in text form, some of which appear to be of minor importance. Furthermore, the\nfigures are not always clear, with key distances or interactions discussed in the manuscript being sometimes hard to locate\nin the corresponding figure. Overall, this technically impressive work lacks the clearly stated core message and easily\nidentified conceptual advance that I would expect from a manuscript submitted to a general readership journal. I provide\ndetailed comments below.\nMajor points:\n1. As stated above, the lengthy discussion of structural details in the Results reads as a text summary of the PDB files, with\nlittle sense of the significance of many of the listed distances or contacts. However, this is not uniformly true, as the\ndiscussion of redox-dependent changes in the T1Cu and T2Cu centers is related back to mechanistic details in an\ninteresting way. I wish the rest of the structural discussion was also firmly rooted in mechanistic aspects that are clearly\nexplained to the reader. For example, the mechanism of the enzyme is never illustrated. In my opinion, this should be a\ncore figure in the main text, possibly figure 1. The authors state on lines 116-117 that “These observations collectively indicate\nthat the binding-before-reduction branch of the proposed “random sequential mechanism” predominates during turnover.”,\nbut this mechanism and the associated open question is never shown or further clarified. This is just one suggestion to\naddress the general sense that manuscript describes a number of impressively high-resolution structures at a level of\nuncontextualized detail that is difficult for readers to follow.\n2. The authors emphasize in several places the special importance of using SHELXL for refinement. Their point about using\nunrestrained refinement and estimated standard uncertainties (ESUs) to determine the protonation states of carboxylic acids\n(starting on line 430) is a nice illustration of a unique capability of SHELX, but it is not fully explored. Unrestrained\nrefinement presumably provided ESUs on all distances, so why not use the parenthetical error notation used in this section\nto provide ESUs for all of the reported distances in the manuscript? As just one of many potential examples, on line 369, the\nauthors state that the N-O distances are approximately equal (~1.27 Å). This would be a good place to use those ESUs to\ndirectly show that these bonds are equal to within 1-2 ESUs. Related to my suggestions in point 1 (above), there are several\nopportunities to more thoroughly analyze and better integrate the ESUs into the core scientific argument of the manuscript.\n3. Related to point 1 above, a concluding summary figure that ties together the large amount of information in this manuscript\nwould be very helpful for the reader. Points to address include: the important lessons learned by comparing reduced vs.\noxidized structures, the key differences or similarities between the three species of enzyme studied, and the mechanistic\nimplications of this study.\n4. The authors might consider commenting of the potential influence of 100K data collection used here, which has been\nshown in a large and growing body of work to sometimes distort functionally relevant disorder/dynamics. Because one of the\nprimary advantages of XFEL crystallography is the absence of radiation damage in room-temperature datasets, this point\nmight be particularly important to mention.\n5. The authors might also consider a deeper analysis of the anisotropic atomic displacement parameters (ADPs) that they\nwere able to refine for these high resolution structures. For example, do preferred directions of atomic motion in the active\nsite differ between these enzymes from different species, in different redox states, or at different pHs? Another possibility is\nto investigate whether the ADPs relate to observed disorder for the bound NO2-, or if there is there any indication of how the\ntop-hat vs. side-on conformations of NO2- differ in atomic displacements.\nMinor points.\n6. SHELXL is misspelled in multiple places and there are other typos that should be corrected.\n7. Referencing to figures in the text would benefit from specifying panel, not just figure number.\n8. Owing to the number of different proteins and states characterized, references to which protein and state are being\ndiscussed should be made clearer throughout (e.g. lines 222-227).\n9. Abbreviations are not always defined, e.g. ET is never clearly defined as “electron transfer”.\n10. Some of the water molecules near the T2Cu active site are reported to be very close (e.g. 2.16 Å on line 216). The\nauthors speculate that perhaps one is an OH- anion. Would that permit these close distances? They still seem unexpectedly\nnear to each other and it would be valuable to know the authors’ thoughts on this. Were they identified as clashes during\nvalidation?\n11. As above, the discussion of the spectroscopic data would be better integrated into the manuscript if mechanistic context,\npreferably in the form of one or more figures, were provided. As it is, these data seem more supplemental than central to the\nmanuscript.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Responses to reviewers comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript by S. Samar Hasnain and colleagues is one of the first comprehensive studies of\ncopper nitrite reductases, examining different oxidation states, and substrate binding at different\npH values using X-ray energy. The atomic-resolution structures presented here provide valuable\ninsights into the substrate-binding mode and the coordination of the two copper sites, which are of\ngreat importance to researchers working with nitrite reductases and other copper metalloproteins.\nThe ability to attain information on resting and substrate-bound states also makes this study\nrelevant to those working with enzymes. The manuscript presents a substantial amount of new\ndata and is generally well-written and clearly presented. The number and quality of figures\nare appropriate for the scope of the study.\nHowever, there are several issues that require clarification or correction before the manuscript can\nbe considered for publication.\nNote: As the manuscript lacks page numbers, all comments below will be referenced to line\nnumbers.\nResponse: Revised manuscript has been page and line numbered.\n1. The abstract should be revised to present the main conclusions obtain in this study.\nResponse: We have removed the generalised sentence “These structures, free from radiation\ninduced chemistry with SHELXL refinement, have provided unprecedented insight into redox\nenzymes mechanisms.” by “A consistent picture has emerged with the observation of a penta-\ncoordinated catalytic type 2 Cu centre in the resting state in all cases. A tetra-coordinate reduced\nCu site with single water has been observed for the first time giving structural support to the\nrandom-sequential scheme.”\n2. Line 50: Role in Nitrification: The authors state that the enzyme is a key enzyme in the\nnitrification pathway. To the best of my knowledge, this enzyme is primarily involved in the\ndenitrification pathway. I would appreciate a clarification and appropriate referencing on how the\nenzyme fits into nitrification, or a correction if this was a misstatement.\nResponse: Though the bulk of research has focussed on its involvement in denitrification and\nnitrifier denitrification pathways. However, in recent years a CuNiR has been characterized from\nan anammox organism (energy generation involving the anaerobic conversion of ammonium to N\n2\nvia nitrite). In the context of nitrification, the discovery that NO is produced by hydroxylamine\nreductase as an obligate intermediate in the first stage of nitrification, a role for NirK in catalysing\nthe oxidation of NO to form nitrite has been proposed (JD Caranto & KM\nLancasterpnas.org/cgi/doi/10.1073/pnas.1704504114). Consistent with this, nirK is present in all\npublished genomes of ammonia-oxidizing archaebacteria including those which do not denitrify,\nstrongly suggesting that it participates in multiple several steps of the nitrogen cycle.\nReference 3 is not appropriate to support this sentence. Please revise your references throughout\nthe manuscript.\nResponse: This was an error. It has been replaced by references 6-8 including our recent review.\n3. Lines 56 to 70: Missing references. This section includes several assertions and background\ndetails that should be supported by citations. Please provide appropriate references to substantiate\nthe statements made here.\nResponse: We had mentioned references 6-12 on line 56. We have now broken it down and\nprovided additional references going up to 17.\n4. Classes of Nitrite Reductases –The different classes of nitrite reductases used in this study are\nonly introduced later in the manuscript (Line 395). For clarity and better flow, I strongly suggest\nthat the authors introduce this classification earlier, ideally in the Introduction or at the beginning\nof the Results section.\nResponse: We did introduce different classes very early on in the Introduction. On Lines 52-54, we\nsaid “Two distinct classes of nitrite reductases are found in denitrifying organisms, a cytochrome\ncd type, encoded by nirS and the more widely distributed copper-containing enzymes encoded\n1\nby nirK6,7” and in the context of CuNiRs sub-classes, we mention blue and green CuNiRs on Line\n58. We have added it also at line 177.\n5. Methods: The Methods section lacks essential details about the protein samples used for\ncrystallization. In particular, the authors should provide more information on the metal content of\nthe proteins, including the number of copper atoms bound per polypeptide chain. Additionally, data\non protein purity, such as absorbance ratios, and the specific activity of the enzyme should be\nreported to confirm that the crystallized protein was functionally competent. The occupancy of the\ncopper site obtained from structural refinement should also be included in the manuscript text, as\nthis information is important to support the structural interpretation of the active site.\nResponse: We added references to relevant papers, T2Cu occupancy we added to table S3.\nBrjNiR was purified as previously reported [Metallomics, 12(2020):2084–2097; Archives of\nBiochemistry and Biophysics, 770 (2025):110467]. The Cu content was determined using either\nthe Biquinoline method or atomic absorption spectrometry. A ratio of 2 mol of Cu to 1 mol of BrjNiR\nmonomer is usually obtained after SEC, with a minimum of 1.9. Another checkpoint is the A /A\n280 592\nratio, but this depends on the oxidation state of the T1Cu centre. Good, as-purified samples usually\nyield a ratio of 16–18. To check the integrity of the T1Cu and T2Cu centres, the samples were\nsubjected to EPR analysis. Activity assays were performed using MV, yelding a value 15 mol\nnitrite min-1 mg-1 monomeric BrjNiR. Specific activity of AcNiR and BrNiR has been reported in\n(Daisuke et al), AcNiR/ BrNiR activity is 924/41 nmol s-1(nmol of protein)-1, respectively. The\nsamples used for crystallisation have similar activity. Both samples were fully Cu loaded.\n6. Line 155: Bound hydroxide at low pH. The manuscript reports a bound hydroxide ion at pH 4.8,\nyet it does not offer an explanation for the presence of OH⁻ at such an acidic pH. This observation\nis unusual and warrants further discussion, particularly with regard to the possible involvement of\na nearby residue that might change the local pKa, or to stabilization through metal coordination.\nCan the authors mention the occupancy to identify it was an oxygen atom.\nResponse: We do discuss this and say that “the proximal position of the catalytic Aspartic residue\n(Asp ) is also present, hydrogen bonding to W1 ligand”. We have rephrased it to clarify further.\nCAT\n7. Line 188: Coordination of T1Cu BrNiR. In the discussion of the T1Cu center in BrNiR, the authors\nmention that Met145 adopts two positions in the structure solved at pH 7.3 and in the nitrite-bound\nform. However, no mechanistic or structural rationale is provided for this dual conformation.\nAlthough they cite literature (line 189-190) linking the axial methionine's position to the reduction\npotential of the T1Cu sites, they do not directly relate this to the properties of their own proteins.\nFurthermore, such difference in geometry of the center should be observed in its spectroscopic\nfeatures, such as visible absorption spectra, visible CD or EPR spectra. The visible spectra\nobtained from the crystals should be better analyzed to corroborate this observation. Moreover,\nthe authors should mention whether this effect has been observed in the structures of other T1Cu\nproteins.\nResponse: We had avoided commenting on this in view of partial occupancy but now have included\noptical spectra for both Rhizobia CuNiRs at high pH also. Only in Br2DCuNiR, which shows dual\nconformation of Met145, optical band at 590nm shows a significant reduction in intensity. For\nBrJNiR, the optical spectra at two pH are indistinguishable. We have included this in the text.\n8. Line 425-427: Catalytic Mechanism.The reviewer can agree that, in the mechanism of these\nenzymes, nitrite binds before the electron is delivered, so that it binds to the oxidized T2Cu center\n(with or without T1Cu center reduced). However, the reviewer disagrees with the authors'\nexplanation and interpretation of the spectroscopic data. The authors claim that the low recovery\nof the T1Cu absorption features means that nitrite has to bind before T2Cu is reduced. However,\nthe recovery of the T1Cu absorption features depends on the relative amounts of the reducing\nagent and nitrite, as well as on the catalytic rate constant. The time at which the spectra were\ncollected after the addition of nitrite is also a factor. Therefore, to support their hypothesis of “nitrite\nbinding-before-reduction”, the authors could perform the reaction in reverse order (add nitrite to\noxidized NiR and then dithionite) and collect spectra at the same time points as before.\nResponse: We appreciate reviewer’s suggestion of performing the reaction in reverse order.\nHowever, we have shown (PNAS 2022) in crystal through MSOX studies of oxidised and nitrite\nbound oxidised enzyme that electron transfer from T1Cu to T2Cu occurs only when nitrite is bound\nto T2Cu. The same has been shown by us in laser-flash photolysis and optical spectroscopy\nexperiments which showed rapid ET from photoexcited NADH to the T1Cu centre but little or no\ninter-Cu ET in the absence of nitrite.\n9. pKa of the bell-shaped curve. The authors study the structure of what they designate as “high”\nand “low” pH. Most low pH falls below or are the optimum pH of these enzymes, with high pH\nabove it. It is important to note the pKas of the pH dependence of the catalytic activity of each\nenzyme is not reported and it would help the interpretation of the data. Additionally, the authors\nfail to establish a clear correlation between the protonation state of AspCAT and HisCAT during\ncatalysis. Form their data AspCAT only becomes deprotonated after nitrite binding and at high pH,\nwhereas HisCAT is always protonated as required for activity. Their structures would correspond\nto the optimum pH structures, but further data on proton transfer is not made.\nChange low pH to optimum activity pH\nResponse: We have replaced low and high pH from the sub-headings and given the actual pH\nvalues for the structures.\n10. Low pH structures. At this pH, is the protein damaged? Is the stability of these enzymes high\nat such a low pH? Line 497: Are these low pH values the optimum pH of the enzymes under study?\nResponse: CuNiRs are located in the periplasm and are generally robust with regard to\ntemperature and extremes of pH covered by the pH-range here. ,. For AxNiR the decrease in\nactivity at pH values below the optimum was not due to instability or denaturation of the enzyme\nsince all reactions were linear during the assays down to pH 4.2. (Abraham et al Biochem. J. (1997)\n324, 511- 516). Lower pH values structures are at pH close to the optimum activity pH (pH6).\n11. Use of HEPES | MES. The authors mention several times in the Methods section to have used\nHEPES pH 5.0 or 5.5, this is a typo for sure, as the pKa of HEPES is around 7.5 and at such low\npH it does not have buffering capacity. So, in which conditions HEPES was used?\nResponse: There is no mistake. HEPES solution for crystallisation and soaking of BrNIR crystals\nwas acidified by HCl from 6.8 to a pH of 5.5. As is always the case, buffered protein was centrifuged\nprior to crystallisation.\nFigures and Figure Legends. In the Figure legends add for all cases the indication of which pH the\nprotein was crystallized (including Figure 5).\nResponse: This has been done.\n1. Line 200: Legend of Figure 2. Mention in the Figure legend the meaning of BW. As far as I can\nsee GK in not in this figure. The figure legends need to be revised.\nResponse: GK and P conformations of Asp are clearly marked in panel f. Abbreviations have been\nexplained.\n2. Line 299: Legend of Figure 4.Panel (b) seems to still show the two conformations in cyan and\nblue. Was this done on purpose?\nResponse: Yes. The cyan conformation represents the fully reduced tri-coordinate Cu(I) site where\nflipping of the Ile252 residue is observed.\n3. Line 390: Legend of Figure 6.The Figure legend mentions LD, which is not in the figure. Please\nindicate the meaning of water WG and GK.\nResponse: LD has been replaced by GK. Abbreviations has been explained.\nOther corrections.\n1. Page 46, The c as in cytochrome c should be in italic (please check the text for other\noccurrences). DONE\n2. Page 53, cd1, 1 is subscript and not in italic. DONE\n3. Line 353, indicate at which pH the protein was crystallized. DONE\n4. Line 401: japonicum without capital letter. DONE\n5. Line 438-440: sentence needs to be revised. DONE\n6. Line 453-456: “… allowed us to conclude that these residues …” residue or residues, seems to\nbe related only to HisCAT. CORRECTED\n7. Line 485 (and other below): penta-coordinate substitute for penta-coordinated. DONE\n8. Line 485: revise “tris-his coordination and two T2Cu ligands” – not usual designation and missing\nwords?\nResponse: Changed to “T2Cu(II) to be penta-coordinate, with a (His) coordination and two solvent\n3\nligands, either two waters or a water and a hydroxide.”\n9. Line 486: and a hydroxide (see above point 8)\n10. Line 558 (and other): microL or mg/mL (correct the Liter as capital letter). DONE\n11. Line 675: concentration and buffer.The designation microm, is a typo for microM? DONE\nThe buffer used was really HEPES pH 6.5? It would be on the limit of the buffering capacity. The\nspectra were collected after nitrite addition to the reduced protein at which time points? This should\nbe mentioned in the Figure legend. The same should be mentioned for the oxidized form incubated\nwith nitrite. Please check my comment above. We have addressed this under crystallisation section\nof the methods. See response to point 11 above.\n12. Line 686: to be removed?\nDone\n13. References. The reference style needs to be revised as it is not homogenous throughout the\ndocument: different fonts used, different formats, missing DOI for most, name of the journals not\nuniform, bold year or volume, date sometimes in parenthesis, titles with capitalized words in some\ncases and in other not, microorganism’s name should always be in italic. DONE. We have not\nadded DOIs.\nReviewer #2 (Remarks to the Author):\nThis manuscript presents, to the best of our knowledge, the first atomic resolution XFEL structures\nof copper nitrite reductases (CuNiRs) in multiple functional relevant states, as oxidized, reduced,\nand nitrite-bound forms. By exploiting high-energy (13 keV) femtosecond X-ray pulses at SACLA\nand refining with SHELXL, the authors achieve structures free from radiation-induced chemistry,\novercoming a major limitation of synchrotron studies where photoreduction distorts the copper\nsites. The work integrates crystallography with single-crystal spectroscopy to confirm the redox\nstate of the type 1 copper site and supports new mechanistic insights into nitrite binding modes,\nwater/hydroxide coordination, and the protonation states of key catalytic residues, such as\nAsp and His . These findings may have broad implications for understanding proton-coupled\nCAT CAT\nelectron transfer, substrate gating and binding and reactivity in CuNiRs. Moreover this manuscript\nrepresents the first application of SHELX, a suite of crystallographic software, to XFEL atomic\nresolutions structures. The manuscript is well-written and the methods described in the main text,\nand the details in the supporting information, are enough detailed to allow reproduction. Overall,\nthe work is exciting and deserve to be published in Nature Communications journal after\nclarification of the following points:\n1) Figure 4 d, e, f are almost certainly type 1 not type 2 Cu sites as they contain cys and met\nresidues. I find it hard to believe that if the coordination number of the copper changes (losing a\nwater) and the copper shifts positions that the imidazoles of the ligands also do not rotate. Figure\n4b implies they don’t. If the authors want to really compare these structures they need to show\nconformations of all atoms at the active site. Figure 4d seems to account for these differences.\nResponse: We do clearly state that in Figure 4, panels d, e and f are for T1Cu site. Panels a to c\nare for T2Cu site where panel b and c show two conformations of reduced state (both deduced\nfrom the single structure of the chemically reduced T2Cu site shown in panel a). Panel a clearly\nshows that imidazoles are single conformation with no smearing of electron density,\n2) Page 18, they are inferring protonation states from bond lengths and angles not determining\nprotonation states. The latter case, actual seeing the protons would require a neutron structure.\nResponse: We agree but are reassured of our assignment of the protonation state, as we have\nshown in 2019 that the protonation state deduced from the high-resolution XFEL crystallography\nstructures of AxNiR in the resting state agrees with neutron crystallography of perdeuterated\nenzyme (Reference 15 “Catalytically important damage-free structures of a copper nitrite\nreductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography).\n3) We get the point that these are “the first” structures. The authors should only state this once and\nthen focus on what their results mean. The reader feels beaten over the head with this emphasis\non novelty when this statement is constantly repeated in abstract, introduction, results and\ndiscussion.\nResponse: DONE\n4) The abbreviation for the bridging water is sometimes inconsistent. On page 7 it is written as\nW (with Br as subscript), while in Figures 2a, 2b, and 2c it is labelled as BW. The notation should\nBr\nbe standardized along the text and figures.\nResponse: Corrected.\n5) As inferred by the authors in the Figure 2 captions, the loop connecting the His ligands of the\nT1Cu and T2Cu sites is proposed to act as a “sensor” in nitrite binding. Given the atomic resolution\nof these structures, it would be nice to know whether the authors compared the conformation of\nthis sensor loop across the different functional states (oxidized, reduced, nitrite-bound). For\nexample, an analysis of the RMSD calculated on the Calfa atoms of this loop could highlight small\nrelevant rearrangements. Including such a comparison may strengthen the argument for its role in\nsubstrate binding.\nResponse: We have done this comparison, but changes are insignificant to be included in the\nmanuscript. We note that despite the atomic resolution, the quality of maps is not as good as what\nwe have obtained at synchrotron storage ring at similar resolution. For example, in AcNiR at 0.87Å,\nsynchrotron X-ray structure, we could identify ~1650 hydrogen atoms compared to ~2700 expected\nhydrogen atoms (see Figures 3 & 4 in IUCrJ (2015). 2, 464–474). There are potentially several\nreasons which need further systematic work to be undertaken, e.g. storage rings are DC stable\nsources while XFEL are pulsed sources where each pulse is a new source with significant\nvariations and multiple crystals are used with each providing multiple spots. We prefer not to\ninclude such comments in the manuscript but are sharing with the reviewer.\n6) How the authors determined the occupancies of residues around the T1Cu and T2Cu centers?\nIt is not entirely clear from the methods. Were the occupancies adjusted manually based on the\nelectron density, or were restraints applied to refine?\nGiven the importance of correctly modeling partial occupancies near the active site, a more detailed explanation in the\nMethods would be very helpful. The inclusion of specific refinement criteria to justify alternate conformations and their\npercentage would increase the strength of the results. In addition, including Fo-Fc omit maps in\nthe supporting information could further support the presence of alternate conformations or partial\noccupancies.\nResponse: Positions of residues with multiple conformations were modelled manualy and assigned\nequal number, for example 50% for double conformations or 30% for side chains with 3\nconformations and then refined by SHELX. Within T1Cu and T2Cu neighbourhoods, occupancies\nof the residues with a single conformation were set to 1.0; We included this in the methods section.\n7) In the BrCuNiR structure at pH 5.5 (Figure 2a), His250 appears to be involved in a different\nhydrogen-bonding network compared to the pH 7.3 structure (Figure 2b). Could the authors clarify\nwhether they observe any difference in the orientation of His250 between these two conditions?\nAdditionally, what is the role of the bridging water at pH 7.3? Does it form new hydrogen bonds\nrespect the structure at pH 5.5? These points should be addressed, as subtle structural differences\nmay provide a structural rationale for the lower catalytic activity observed at neutral pH.\nResponse: At high pH His has stronger bond with W2 (2.9 Å), than with bridging water 3.4 Å,\nCAT\nwhile at low pH the distances are almost equal. We have expanded discussion on changes in\nHis . For example, in lines 167-171, we have written “The catalytic Histidine residue (His250;\nCAT\nHis ) bridges Asp through a conserved bridging water (W ) and its imidazole ring is rotated\nCAT CAT Br\ntowards Glu274, with its Nδ1 atom hydrogen bonding with the carbonyl oxygen of Glu274 at 2.70\nÅ (Supplementary Fig. 3a). This is consistent with the previous SF-ROX structure at 1.3 Å\nresolution16.”\n8) The authors state that all structures were collected at 100 K. However, at cryogenic\ntemperatures protein flexibility, hydrogen-bonding interactions etc. can differ significantly from\nthose occurring in solution under physiological conditions. A short discussion of how cooling may\ninfluence the observed hydrogen-bond network, nitrite binding, and mechanistic interpretations\nwould need to be added.\nResponse: This is a very pertinent comment which we had not addressed. There is always a\nproblem of correlating biophysical studies of enzyme catalysis and how they reflect the reality of\nthe different physiological environments that enzymes are exposed to in vivo. In the case of\nCuNiRs this is reflected in differences between the extent of inter Cu ET in solution and in crystallo.\nNever-the-less our MSOX studies of nitrite-soaked crystals have provided unprecedented insight\nto the detail of structural changes that occur during the reduction of nitrite by CuNiRs in crystals at\ncryogenic temperatures. These MSOX studies have also been carried out at elevated\ntemperatures (>190K), above the glass transition temperature,, permissive for conformational\nchanges or H-bonding interactions to occur. Room temperature MSOX studies have also shown\na consistent picture but of course with limited number of intermediates and rapid loss of resolution\ndue to wider radiation damage. The ability of nitrite crystals to undergo the reduction of nitrite,\nformation of NO adducts and then its loss has been well documented in multiple MSOX studies\n(IUCrJ (2018). 5, 283–292, https://doi.org/10.1016/j.jmb.2024.168706,\nhttps://doi.org/10.1073/pnas.2205664119, etc). In the context of the comment raised by the\nreferee, nitrite was added to the crystal at RT before data collection at 100 K. We have previously\nshown X-ray generated electrons in MSOX studies reduce the T1 centre first and that in the\npresence of nitrite, ET and catalysis occur. We consider that this makes the FRIC structures\npresented here directly relevant to single turnover events during catalysis. This has been clarified\nin the revised manuscript, and we thank the referee for raising this issue.\n9) The primary concern with the paper is not in the results, which are highly significant, but in the\nlack of broader analysis in the discussion. Now that we have all these novel structures, what do\nthey tell us about the catalytic mechanism of the system? We are given exhaustive detail on\nstructures of both type 1 and 2 Cu centers, on the substrate bound forms and on various pH\nconditions. But in my opinion, none of this data is brought together to assess catalytic mechanism\nfor nitrite reduction that have been proposed. For example, is proton gating critical and how is it\nachieved both by changing the catalytic site and also the electron transfer site? Are structural\nchanges observed consistent with known reduction potential changes in these systems based on\nsubstrate binding and protonation? How is the nitrite conformation important for proposed\nmechanisms (they hint at this in Fig 2 results showing gatekeeper conformations but never discuss\nhow this influences the type 1 center structure and hence potential)? I could go on… The authors\nhave really addressed excellently critical structural issues with this work; however, they have\ndropped the ball when tying the importance of their discoveries to the known catalytic system. This\ndiscussion is what is required to elevate this article to the level of Nat Comm, otherwise, the work\nshould be in a more specialized, but still high impact crystallographic journal. With the proper\ndiscussion, I would fully support Nat Comm publication.\nResponse: We have included a scheme outlining the main findings. Both PCET and Gating of\nsubstrate are well documented (references 22-25 and 28) and are indicated on the scheme. The\nscheme highlights a number of new findings including (1) resting state for all of the NiRs being\npenta-coordinate, (2) dithionite reduced despite the complete loss of T1Cu signal, being in two\nstate, one where T2Cu has tetra-coordination with single water molecule that would be capable of\nbinding the substrate and turn-over. The random-sequential mechanism was based on solution\nstudies where dithionite treatment would have been assumed to reduce the T1Cu to fully reduced\nstate due to the loss of colour, (3) at high pH, the bridge between His\nCAT\nand Asp\nCAT\nis lost with\nHis bridging to one of the T2Cu-coordinated solvent ligands providing a structural explanation\nCAT\nfor lower activity at higher pH, (4) at low pH, one of the coordinated waters is quite short suggesting\na potential water – hydroxide (OH-) resting state of the enzymes, consistent with the proposed\nbased on high-resolution neutron crystallography and computational studies53,54.\nMinor issues:\n- Typos along the text like “SHLEXL” in the abstract. DONE\n- Mixing british and american English spellings (e.g., oxidised vs oxidized, modelled vs modeled).\nPlease standardize to one style, in line with the journal guidelines. DONE\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe authors report several atomic resolution crystal structures of copper nitrite reductase (CuNiR)\nfrom Bradyrhizobium and Achromobacter species obtained using an X-ray free electron laser\n(XFEL), providing detailed and radiation damage-free views of the two metal centers in these redox\nproteins. The authors used an approach where they rotate and serially expose multiple regions of\nlarger crystals to the XFEL beam at cryogenic temperature (SF-ROX), which permits using many\nfewer crystals to obtain a complete dataset. SF-ROX has the clearly demonstrated potential to\ngenerate data whose resolution exceeds that typically obtained using more conventional serial\ndelivery of randomly orientated microcrystals typically used at XFELs. The authors have previously\npublished pioneering work on this method and applied it to CuNiR enzymes (PMID: 29354268,\n31316819). Therefore, this is an established method and the principal advance being reported in\nthis manuscript is obtaining markedly improved resolution data for CuNiRs from multiple species.\nThis improved resolution allows them to refine the models in SHELXL, which can produce\nestimated standard uncertainties (ESUs) on model parameters. Overall, this is an impressive\nexample of crystallography at XFEL sources being used to answer questions that are not\neasily accessible using other X-ray sources owing to radiation damage.\nDespite its technical strengths, the central findings of the study are difficult to summarize, as they\ncover the details of the changes in the metal and coordinating residue geometries as a function of\nspecies, pH, and redox state. In support of this impression, the Abstract provides unusually little\nsense of what was learned in the work.\nResponse: Thank you for your positive comments. We have summarised some of the key findings\nin the abstract and Scheme 1.\nWhile the results are discussed in exhaustive detail, it is unclear (at least to this reviewer) what the\ncore general message of the manuscript is. For example, I cannot confidently determine what a\nreader (particularly one who is not actively working on the structural biology of CuNiRs) might take\nfrom this study. This is at least partially the result of a difficult writing style, focused on providing\nlists of interatomic distances in multiple proteins in text form, some of which appear to be of minor\nimportance. Furthermore, the figures are not always clear, with key distances or interactions\ndiscussed in the manuscript being sometimes hard to locate in the corresponding figure. Overall,\nthis technically impressive work lacks the clearly stated core message and easily identified\nconceptual advance that I would expect from a manuscript submitted to a general\nreadership journal.\nResponse: We thank the reviewer. It has encouraged us to revise the discussion and include a\nscheme summarising the new findings pertinent to enzyme mechanism is included.\nI provide detailed comments below.\nMajor points:\n1. As stated above, the lengthy discussion of structural details in the Results reads as a text\nsummary of the PDB files, with little sense of the significance of many of the listed distances or\ncontacts. However, this is not uniformly true, as the discussion of redox-dependent changes in the\nT1Cu and T2Cu centers is related back to mechanistic details in an interesting way. I wish the rest\nof the structural discussion was also firmly rooted in mechanistic aspects that are clearly\nexplained to the reader. For example, the mechanism of the enzyme is never illustrated. In my opinion,\nthis should be a core figure in the main text, possibly figure 1. The authors state on lines 116-117 that\n“These observations collectively indicate that the binding-before-reduction branch of the proposed\n“random sequential mechanism” predominates during turnover.”, but this mechanism and the\nassociated open question is never shown or further clarified. This is just one suggestion to address\nthe general sense that manuscript describes a number of impressively high-resolution structures\nat a level of uncontextualized detail that is difficult for readers to follow.\nResponse: We have revised the manuscript (Results and Discussion significantly) and included a\nscheme summarising the key findings.\n2. The authors emphasize in several places the special importance of using SHELXL for\nrefinement. Their point about using unrestrained refinement and estimated standard uncertainties\n(ESUs) to determine the protonation states of carboxylic acids (starting on line 430) is a nice\nillustration of a unique capability of SHELX, but it is not fully explored. Unrestrained refinement\npresumably provided ESUs on all distances, so why not use the parenthetical error notation used\nin this section to provide ESUs for all of the reported distances in the manuscript? As just one of\nmany potential examples, on line 369, the authors state that the N-O distances are approximately\nequal (~1.27 Å). This would be a good place to use those ESUs to directly show that these bonds\nare equal to within 1-2 ESUs. Related to my suggestions in point 1 (above), there are several\nopportunities to more thoroughly analyze and better integrate the ESUs into the core scientific\nargument of the manuscript. Response: We have done this throughout the Results section and\nDiscussion. We have also included Supplementary Tables 2 and 3 with these details.\n3. Related to point 1 above, a concluding summary figure that ties together the large amount of\ninformation in this manuscript would be very helpful for the reader. Points to address include: the\nimportant lessons learned by comparing reduced vs. oxidized structures, the key differences or\nsimilarities between the three species of enzyme studied, and the mechanistic implications of this\nstudy.\nResponse: We have included Scheme 1 as per suggestion. We are grateful to the reviewer for\npointing this shortcoming.\n4. The authors might consider commenting of the potential influence of 100K data collection used\nhere, which has been shown in a large and growing body of work to sometimes distort functionally\nrelevant disorder/dynamics. Because one of the primary advantages of XFEL crystallography is\nthe absence of radiation damage in room-temperature datasets, this point might be particularly\nimportant to mention.\nResponse: We have added this in the concluding paragraph “We conclude that the ability to obtain\natomic resolution damage free structures for a variety of functionally relevant states using few (50)\ncrystals of ~ 30 m thicknesses by deploying 13keV X-rays from an XFEL in conjunction with the\nSF-ROX methods holds a promise for elucidating enzyme mechanism for many metalloenzymes.\nSF-ROX method, like SFX, is also applicable for ambient temperature crystallography but the\npropagation length for damage resulting from the first femtoseconds pulse is going to be\nsignificantly larger thus requiring a much larger number of crystals compared to those used here.\nThe extent of propagation that may occur at room temperatures requires a systematic study. ”\n5. The authors might also consider a deeper analysis of the anisotropic atomic displacement\nparameters (ADPs) that they were able to refine for these high resolution structures. For example,\ndo preferred directions of atomic motion in the active site differ between these enzymes from\ndifferent species, in different redox states, or at different pHs? Another possibility is to investigate\nwhether the ADPs relate to observed disorder for the bound NO2-, or if there is there any indication\nof how the top-hat vs. side-on conformations of NO2- differ in atomic displacements.\nResponse: We have included supplementary figure 4, showing ellipsoidal models of the T2Cu\nactive sites in these structures.\nMinor points.\n6. SHELXL is misspelled in multiple places and there are other typos that should be\ncorrected. DONE\n7. Referencing to figures in the text would benefit from specifying panel, not just figure\nnumber. DONE\n8. Owing to the number of different proteins and states characterized, references to which protein\nand state are being discussed should be made clearer throughout (e.g. lines 222-227). DONE\n9. Abbreviations are not always defined, e.g. ET is never clearly defined as “electron transfer”.\nDONE\n10. Some of the water molecules near the T2Cu active site are reported to be very close (e.g. 2.16\nÅ on line 216). The authors speculate that perhaps one is an OH- anion. Would that permit these\nclose distances? They still seem unexpectedly near to each other and it would be valuable to know\nthe authors’ thoughts on this. Were they identified as clashes during validation?\nResponse: We have included a Fo-Fc OMIT map (Supplementary Fig. 1b), which when compared\nto supplementary Figure 1(a) persuades us that both W1 and W2 exists. It is possible that at high\npH W1 may be split into two but have left it as a single W1 to avoid over interpretation. W2 has\nonly half occupancy. There were no clashes in validation report.\n11. As above, the discussion of the spectroscopic data would be better integrated into the\nmanuscript if mechanistic context, preferably in the form of one or more figures, were provided. As\nit is, these data seem more supplemental than central to the manuscript.\nResponse: The optical spectrum is incorporated in the presentation of the results throughout the\nmanuscript (lines 173-176, lines 220-228, line 244, line 301, line 405, line 425 etc). We hope that\nthe reviewer would be agreeable to keep it in the main text.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse to remaining points of Reviewer 1.\nThe authors have thoroughly revised the manuscript attending to all the suggestions of the\nreviewers. The manuscript has been improved considerably, specially by the inclusion of final\nscheme. Some minor considerations:\nResponse: We appreciate that reviewer is satisfied except for minor points which have\nresponded in detail so that the Editor can be persuaded to accept the manuscript without\nfurther reiterations.\n1. Though not pointed out before, panel 1c is not really a figure and the reviewer suggests\nmoving it to supplementary materials.\nResponse: We have removed Figure 1C and included it in the supplementary material.\n2. Figure 3: pH at which the experiment was performed should be stated in the figure legend\n(for the crystals and solution experiments).\nResponse: We have included this information.\n3. Relative to this experiment, this reviewer does not agree with the statement that low\nrecovery of absorbance when nitrite is added to the reduced enzyme, means that it is because\nnitrite does not bind to the reduced catalytic center. In the literature, similar experiments\nperformed with other CuNir from other organism clearly show a complete or nearly complete\nrecovery of the T1Cu absorption bands after 10 min. Either these enzymes have low catalytic\nrate constant or low T2Cu center occupancy upon reduction (might be explained by the values\nin table S3). See publications: 10.1039/d0mt00177e and 10.1016/j.bbagen.2017.10.011.\nResponse: We DO NOT say that nitrite does not bind to the reduced enzyme. We say reactivity\nwith reduced enzyme is sluggish. Also, one of the authors (Ferroni) of the current paper is a\nlead or a corresponding author of the above papers which the reviewer cites. In addition, in\nexperiments that either the nitrite binding is limited when T2Cu is reduced or that the T1Cu-\nT2Cu ET is affected. There is substantial literature that support the observation that reduced\ntype 2 Cu site are unable to bind or weakly bind nitrite or inhibitory ligands such as azide, and\nto that reduce T2Cu site react very sluggishly with nitrite leading to only a slow re-oxidation of\nthe type 1 centre (J. Mol. Biol. (1999) 287, 1001; J Biol Chem (1997), 272, 28455). In last of\nthese papers (JBC) structure of nitrite bound to AfNiR with low occupancy of nitrite. For further\ndiscussion on pH aspect see response below.\nWe have modified the text to say, “The low recovery of T1Cu optical spectrum (~20 %)\nindicates that binding of nitrite to the reduced T2Cu(I) site is limited but not prevented,….”\nAnother explanation could be the pH at which the experiment was performed, as at higher pH\nthe activity of the enzyme decreases (buffer pH change with temperature). Can the authors\ncomment on this.\nResponse: As suggested by the reviewer, this is a real issue for solution studies (see A\nTemperature Independent pH (TIP) Buffer for Biomedical Biophysical Applications at Low\nTemperatures (Chem Commun 2008, 7, 823–825. doi:10.1039/b714446f). We used Tris/HCL,\nHEPES and Citrate buffers, all of which will become significantly more acidic at\ncryotemperatures in solution.\nIn the case of flash-freezing of crystals of BrNiR, as in our current paper, we have shown in\nour multiple MSOX studies that enzyme turnover at pH 5.5 is not affected (PNAS 2022\npnas.2205664119, j.jmb.2024.168706, Chem. Sci., 2020, 11, 12485–12492, etc). We have\nshown in optically validated MSOX studies that PCET from T1Cu to T2Cu is disrupted resulting\nin a significantly slower conversion of substrate (j.jmb.2024.168706). In these papers we have\ndemonstrated that nitrite can be soaked to full occupancy and that metal sites have mostly full\noccupancy of Cu. Solution metal analysis in general are not specific to a site but to the overall\nmetal content.\nThis reviewer just does not agree with sentence in line 129-130 | 495-496 – “one branch of\nthe random sequential mechanism predominates relative to the other”, taken from the\nexperiment shown in Fig.3.\nResponse: This is a position appears to be a conviction. Our response is simply against such\na belief.\n4. Some typos are still found in the manuscript but will for sure be corrected at a later stage of\nthe manuscript.\nResponse: We have gone through the manuscript carefully again."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoroughly revised the manuscript attending to all the suggestions of the reviewers. The manuscript has\nbeen improved considerably, specially by the inclusion of final scheme. Some minor considerations:\n1. Though not pointed out before, panel 1c is not really a figure and the reviewer suggests moving it to supplementary\nmaterials.\n2. Figure 3: pH at which the experiment was performed should be stated in the figure legend (for the crystals and solution\nexperiments).\n3. Relative to this experiment, this reviewer does not agree with the statement that low recovery of absorbance when nitrite is\nadded to the reduced enzyme, means that it is because nitrite does not bind to the reduced catalytic center. In the literature,\nsimilar experiments performed with other CuNir from other organism clearly show a complete or nearly complete\nrecovery of the T1Cu absorption bands after 10 min. Either these enzymes have low catalytic rate constant or low T2Cu center\noccupancy upon reduction (might be explained by the values in table S3). See publications: 10.1039/d0mt00177e and\n10.1016/j.bbagen.2017.10.011.\nAnother explanation could be the pH at which the experiment was performed, as at higher pH the activity of the enzyme\ndecreases (buffer pH change with temperature). Can the authors comment on this.\nThis reviewer just does not agree with sentence in line 129-130 | 495-496 – “one branch of the random sequential\nmechanism predominates relative to the other”, taken from the experiment shown in Fig.3.\n4. Some typos are still found in the manuscript but will for sure be corrected at a later stage of the manuscript.", "reviewer_2": "The authors have addressed all my comments satisfactorily, and I now consider the manuscript suitable for publication in\nNat.Comm.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "The authors have addressed my concerns from the initial review. This manuscript is much-improved and clarifies key points\nthat were missing before.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have thoroughly revised the manuscript attending to all the suggestions of the reviewers. The reviewer has no\nfurther comments."}, "author_response": ""}]

Decision letter