Peer review of: Redox-coupled proton pumping drives carbon concentration in the photosynthetic complex I (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The work entitled “Redox-Coupled Proton Pumping Drives Carbon Concentration in the 
Photosynthetic Complex I “ presents a structural study which combines CrioEM data, with 
Structural modeling, Molecular Dynamics (MD) and QM based calculations to analyze several 
important mechanistic aspects of structure function relationships in photosynthetic complex I. The 
results reveal a novel catalytic carbonic anhydrase module (different form alpha and beta carbonic 
anhydrases), with a unique CupA active site harboring a Zn 2+ coordinate to His and Arg residues 
and proposed to be connected thorugh proton exchange/transfer to the proton-pumping modules 
which are coupled by a 200 Å electrostatic wire to the electron transfer module of the enzyme. A 
proposed CO2 channel connecting the protein boundary with the Zn active site is also proposed 
and several mechanistic conclusions are drawn based on a interpretation of the structure and 
molecular simulation results. 
Although the work is novel, relevant and very interesting, it falls short on many technical aspects 
(particularly related to the molecular simulations) which prevent publication in its current form. 
Also, presentation should be improved since many important details have been left out in the main 
text which are important and necessary to understand and really gauge the impact of the work. 
Therefore I suggest publication only provided major revisions are performed and simulation 
aspects improved, Specific points follow: 
 
 
The pargraph beginning with “ To probe the catalytic properties of the CupA module, we performed 
quantum chemical calculations on the CO 2 hydration reaction and compared this to the reaction 
energetic in aCA and bCA (Fig. 2b, Extended Data Fig. 5, see Methods)... “ needs lots of work. 
First, in the main tecxt the authors give no detail on the performed calculations, just mentioned 
that they used QM/MM methods. Authors should properly described the level of theory, and the 
type of calculation, particular in relation as to how the energy barriers were obtained. 
Moreover, some brief explanation of the reaction mechanism for ll three enzymes bCA, aCa and 
CupA is needed in order to proper understand the differences. Key elements are missing, such as 
who is the key residue acting as base, that removes the proton from the Zn coordinated water that 
is supposed to initiate the reaction. 
 
Figure 2, needs more explanation. In particular concerning the energy diagram. Authors write DG, 
however they only computed “energies” with entropic corrections using single points?. Therefore, 
the profiles are not actual G but E+ZPE. To really compute free energy profiles authors need to 
perform QM/MM dynamics with some free energy (or PMF) determination method such as Umbrella 
sampling, Metadynamics or MSMD. 
 
Authors need to provide further detail on all three computed reactions. bCA, aCA and CupA. In 
supplementary data only the profile for CupA step 1 is shown, no data is provided for bCA and aCA 
and the remaining steps of CupA. Authors MUST provide free energy (or E+ZPE profiles if they 
want) of all steps in all three enzymes. Moreover, authors should clearly explain the reaction 
coordinate used in each reaction step and describe initial conditions. 
 
Also important, since the reaction involves proton transfer. The authors start with Y41 
deprotonated, but who takes the proton from Y41?. Who acts as base in a/bCA??. Also related to 
Y41 and pka calculations (Figure 5F from extended data). Pka was computed using a continuum 
model which is not really accurate, authors should actually compute the pKa using better methods 
such as constant pH Molecular dynamics (Se Dr Roibergs works on the subject). Why do they 
report two pkas??? 
 
Concerning Extended data Figure 6. Authors performed MD simulation and looked at the presence 
of water molecules which they claim show potential proton transfer wires?. However no 

quantitative measure of the solvation is given. Authors could compute key position of water 
molecules inside the protein using IFST method, i.e determining the Water Sites and their 
properties (see for examples works by Gauto et. al. and/or WATCLUST method doi: 
10.1093/bioinformatics/btv411). 
 
Finally, authors claim that protein hosts a tunnel that concentrates CO2. However, they only 
provide anecdotal evidence of CO2 moving inside the proposed tunnel. Authors should compute 
the free energy profile of CO2 migration along the tunnel to really measure how much the tunnel 
contributes (in relative amount) to concentrate CO2 inside the protein. See for example how this is 
performed by Myglobin to concentrate O2 inside the active site doi: 10.1074/jbc.M112.426056) 
 
Marcelo A. Marti 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in 
energy metabolism. In humans, defects in complex I are linked to severe neurodegenerative 
diseases. During the last years several sub-families of energy converting NADH:ubiquinone 
oxidoreductase have been identified that evolved from each other and that share a similar 
structure and a related mechanism. Cyanobacteria and plants contain an energy-converting 
ferredoxin:plastoquinone oxidoreductase consisting of several subunits with most of them being 
related to those of mitochondrial complex I. Very recently, the structure of this enzyme complex, 
also called photosynthetic complex I, from Thermosynechococcus elongatus was determined by 
means of cryo-electron microscopy (cryo-EM). 
 
The manuscript by Schuller et al. describes the cryo-EM structure of the NDH-1MS isoform of the 
Thermosynechococcus elongates photosynthetic complex I. This isoform is equipped with an 
additional module catalyzing a carbonic anhydrase (CA) reaction. The authors describe the 
ferredoxin and the plastoquinone binding sites and several lipids bound to the enzyme complex. 
Most importantly, they demonstrate that the CA domain is made up of CupA and S on the 
cytoplasmic side of the membrane. The authors unequivocally show that this domain contains a 
catalytically active Zn(2+) ion that is ligated in a different way compared to the well-known α and 
ß CAs leading to a different type of mechanism. The proposed mechanism is substantiated by 
theoretical energy calculations. It is nicely argued that proton translocation by the photosynthetic 
complex I facilitates the CA reaction by removing the proton, a reaction product. Furthermore, a 
mechanism is provided that could explain the propagation of the proton translocation along the 
membrane part of the complex. 
 
The manuscript is very well written, easy to follow and contains novel and highly interesting 
information. The experiments and the data are sound and well documented. The manuscript 
benefits very much from the impressive interplay of structural analysis and topical theoretical 
methods. I just have a few minor points to possibly be considered: 
 
Page 2, third para, first sentence: ‘The 0.5 MDa complex has an overall U-shape with 18 isolated 
subunits.’ However, in Extended Data Table 2, 19 subunits are listed. 
 
Page 3, first para, first sentence: ‘The CO2-concentrating CupA/S module (CO2 uptake/CO2 
hydration protein, ChpY)….’ ‘up’ is underlined but does not contribute to the abbreviation. The 
abbreviation reads as CO2 hydration protein, maybe a better wording can be found in order to not 
confuse the non-experts. 
 
Page 3, second para, last sentence: Is there any evidence that the chlorophyll α/ß-carotene play a 
role in light-induced regulation? If not, I would delete the sentence in the main text and maybe 

place it as pure speculation in the legend of the corresponding figure. 
 
Extended Data Fig. 2: I would propose to also label the subunits above NdhF3 in 2a. It should be 
explained in the legend why NdhN and M appear twice in 2c/e. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Cyanobacteria contain a set of enzymes related to respiratory complex I, the NDH-1 family, with 
diverse functions that appear to have evolved to deal with the challenges of oxygenic 
photosynthesis. A number of papers appeared earlier this year that showed the structure of NDH-
1L, which is similar to respiratory complex I, but lacks the first three subunits of this complex that 
transfer electrons from NADH to the quinone binding site. Instead, a number of specific subunits 
allow electron transfer from ferredoxin. The paper by Schuller et al. concerns the cryo-EM 
structure and molecular dynamics studies of NDH-1MS from Thermosynechococcus elongatus, an 
enzyme involved in carbon concentration. The authors show that NDH-1MS contains a 
noncanonical carbonic anhydrase module, located at the end of the proton transfer chain, and 
develop a mechanism for its function in concentration CO2. 
 
The combination of cryo-EM, model building and molecular dynamics studies yields new insights in 
cyanobacterial photosynthesis that will be of high interest to experts in the field. The paper is 
overall well-written; it would however benefit from some modifications, in particular concerning 
the figures and figure legends, as detailed below. 
 
Specific points: 
1. p. 3, assignment of the zinc ion in CupA: the cryo-EM density for this ion and its coordination 
should be shown in a supplementary figure. The zinc ion should also be shown in figure 3a. 
Further, the text states that the assignment is “supported by multi-element analysis data 
(Extended Data Fig. 2e)”. However, this figure does not show this. There is mention of Zn in 
Extended Data Fig. 2d, but this is “Inductively-Coupled Plasma Optical Emission Spectrometry”. It 
is totally unclear what this table (2d) shows, and neither this technique nor multi-element analysis 
are mentioned in the Methods. Further, it is stated that the Zn ligand Arg135 has a pKa <7, but 
Extended Data fig. 5f states pKa <0. 
 
2. p. 3 third paragraph, the sentence starting “a non-polar tunnel…” is ungrammatical and can’t be 
understood. 
 
3. p. 4 “global dynamics inferred from the cryo-EM map” would be clearer as “global dynamics 
inferred from the local resolution of the cryo-EM map”. The legends of Ext. Data Fig. 7, which show 
this, are inadequate and should state more explicitly what the color scheme represents. 
 
4. p. 4 “charged residues in the broken helices TM7 and TM12 (Fig. 3)”. It is not mentioned which 
subunit these helices belong to. Figure 3d shows these helices in all three antiporter subunits 
NdhF3, D3 and B. It would be better to state this in the text and refer to figure 3d instead of figure 
3 in full. 
 
5. p. 5 “coupled protonation and/or conformational changes at the NdhF3/NdhD3 interface could 
close the gas channel and decouple the pump…”. This part is very speculative. It is not clear why 
the reverse reaction would close the channel that is presumably always open in the forward 
reaction. 
 
6. Figure 1: This figure shows the cryo-EM map of the protein with a simulated, full atomic model 
of the membrane. Experimentally determined lipids (as seen in Ext. Data Fig. 3) are however not 
shown. The atomic model distracts from the protein map and partially obscures it (e.g. NdhL). It 

would be better to show the EM density map by itself, including observed non-protein density. 
Further, panel c does not show a back view, but a side view. It may be a good idea to reverse 
panel a and c, as a has the same orientation as the model in d, which would then be next to it. In 
panel c, it is not clear what subunit forms the horizontal surface helix (brownish) below NdhK. 
 
7. Figure 3: presumably the CO2 channel is shown in purple in a, b and c, but this is never stated. 
It looks very different in b and c, how was the surface determined? Also the scale and orientation 
of these panels differs considerably and it is unclear how they relate to each other. 3d: what is an 
“experimentally-refined structure”? There are no densities shown in figure 3, so referring to 
“further example densities” is strange. 
 
8. Figure 4: This figure could do with better legends to explain the colors and arrows. Why is one 
of the H+ in the lower panel grey? 
 
9. Methods, image processing: “the motion correction algorithm” should be specified as 
MotionCorr2 and the reference added. 
FSC does not stand for “Fourier shell correction” but “Fourier shell correlation.” 
 
10. Extended Data Fig. 1: twice refinement is misspelled as “refinment”. 
 
11. Extended Data Fig. 3: PGT, SQD and DGD should be defined. It is better not to show 
hydrogens in the atomic models, they clutter the images. 
 
12. Extended Data Fig. 4a: It is unclear what is shown here. Legend for the left panel is missing 
and the right panel does not show the interaction between the subunits, just the surface. 
 
13. Extended Data Fig. 4b: It is unclear what is shown here. Is orange the pdb ID 2MXA, as stated 
in the legends, or the cryo-EM structure? The legend also states that the solution structure is cyan, 
which seems more likely. Further, helix α3, mentioned in the legend and in the text on page 3, is 
not shown. The text mentions movement of α2, α3, the legend α1, α2, α3. 
 
14. Extended Data Fig. 5f: the legend mention “the latter values”. This makes no sense; 
presumably what is meant is the second figure in each column. 
 
15. Extended Data Fig. 6e: it is not clear what is shown here. What color is mouse and what 
cyanobacterial? 
 
16. Extended Data Fig. 7: “Dynamics from cryo-EM resolution map” should be “Dynamics from 
local resolution of the cryo-EM map”.