Photon Factory Activity Report 2016 #34 (2017)

AR-NW12A/2009G578, 2011G519, 2013G504, 2016G657

Atomic Resolution X-ray Crystal Structure Analysis of Pseudoazurin
Takahide Yamaguchi1,2, Kohei Akao1, Akiko Takashina1, Masaki Unno1,2, Robert K. Szilagyi3,* andTakamitsu Kohzuma 1,2*1
Inst. Quantum Beam Sci., Ibaraki University, 2-1-1 Bunkyo, Mito, 310-8512, Japan
2
Frontier Appl. Atomic Sci. Res. Centre, Ibaraki University, 162-1 Shirakata, Tokai, 319-1106, Japan3
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59718, USA1 Introduction
Type 1 copper site of blue copper protein functions aselectron carriers in many biological electron-transfersystems. Therefore, the electronic structure of the coppersite has been studied extensively to elucidate thecorrelation between the structure, unique spectroscopic
properties, and the Cu2+/Cu+ reduction potential. Up tonow, the distortion model describing the presence of axial
or rhombic sites invoked a single copper site. However,the possibility for the simultaneous presence of different
copper sites in the same protein environment has alreadybeen suggested by early resonance Raman measurements
and computational modelling.
Pseudoazurin from Achromobacter cycloclastes(AcPAz) is an electron donor to nitrite reductase andnitrous oxide reductase in the denitrification process. The
previous comprehensive spectroscopic observations
indicated the simultaneous presence of two different Cu2+
sites, and non-covalent weak interaction significantly
influence the ratio. In this experiment, the improvementin crystallization conditions and developments ofsynchrotron beamlines and software evidence the twopositions for the Cu site in AcPAz. The atomic resolution
crystallographic analysis also enabled us to correlate withthe precise computational modelling with our result ofspectroscopy[1, 2].
2 Experiment
The protein of AcPAz was expressed by therecombinant E. coli and purified using ion-exchange
chromatography. The crystal of AcPAz was ontained by
the hanging drop vapor diffusion method. The single
crystal X-ray diffraction data for the AcPAz crystal werecollected at beam line NW 12A (PF-AR). The
temperature of crystal was maintained at 100 K during
measurement. The AcPAz crystal belong to the I222 spacegroup with cell parameters a = 56.1 Å, b = 63.4 Å, c =67.2 Å. The structure of AcPAz was refined at 1.10 Åresolution. (PDB ID: 4YL4)
3 Results and Discussion
The structure of AcPAz at 1.10 Å resolution
demonstrated the co-existence of two Cu position in thesame crystal (Figure 1). The occupancy of Cu positions
was found to be consistent with the population of axial
and rhombic sites from spectroscopic measurements.
Computational modellings using an approximately 6 Åprotein environment around the Cu site for both theoxidized and reduced forms showed that a small scale
inner sphere rearrangement can account for the coexistence of two different redox active sites for amononuclear cupredoxin independently from theemployeddensityfunctionals.
Thestructural
optimizations resulted in an energetically less favourable
axial site by 20 kJ mol-1 than the rhombic, which is inqualitative agreement with the axial site being the minor
and the rhombic site being the major component in AcPAz.
The above discussion for the crystallographic results
and computational modelling led the conclusion that thedual positions of Cu atom in AcPAz can be explained by
the presence of two energetically comparable minima on
the potential energy surface within a highly similar ligand
environment[3]. The small energetic differnece of theseminima indicates the importantce of non-covalent weakinteraction from outer sphere (2nd coordination sphere) in
type 1 Cu site, or other active sites in metalloproteins.

Figure 1. The structure of type 1 copper site ofpseudoazurin with 2Fo-Fc (gray at 1.5 s level) and FoFc(green and red at ± 4.0 s level).
References[1] R. F. Abdelhamid et al., J. Biol. Inorg. Chem. 12, 165(2007).
[2] M. B. Fitzpatrick et al., J. Inorg. Biochem. 104, 250(2010).
[3] T. Yamaguchi et al., RSC Adv., 6, 88358 (2016)* takamitsu.kohzuma.qbs@vc.ibaraki.ac.jp