X-ray crystallographic evidence for the simultaneous presence of axial and rhombic sites in cupredoxins: atomic resolution X-ray crystal structure analysis of pseudoazurin and DFT modelling Authors: T. Yamaguchi, K. Akao, A. Takashina, S. Asamura, M. Unno, Robert K. Szilagyi, and T. Kohzuma This is a postprint of an article that originally appeared in RSC Advances on Sepember 9, 2016. https://dx.doi.org/10.1039/c6ra19282c Yamaguchi, T, K Akao, A Takashina, S Asamura, M Unno, Robert K Szilagyi, and T Kohzuma. "X-ray crystallographic evidence for the simultaneous presence of axial and rhombic sites in cupredoxins: atomic resolution X-ray crystal structure analysis of pseudoazurin and DFT modelling." RSC Advances 6, no. 91 (September 2016): 88358-88365. DOI:10.1039/c6ra19282c. Made available through Montana State University’s ScholarWorks scholarworks.montana.edu X-ray Crystallographic Evidence for the Simultaneous Presence of Axial and Rhombic Sites in Cupredoxins: Atomic Resolution X-ray Crystal Structure Analysis of Pseudoazurin and DFT Modelling T. Yamaguchi,a K. Akao,a A. Takashina,a S. Asamura,a M. Unno, a,b R. K. Szilagyi,c* and T. Kohzumaa,b* Crystal structure analysis of pseudoazurin from Achromobacter cycloclastes (AcPAz) was carried out at atomic (1.10 Å) resolution. The copper ion was localized in two positions at the metal binding site of AcPAz. The occupancies of the copper sites are consistent with the ratio of axial and rhombic signals from EPR spectra and the intensity ratios for blue (axial) and green (rhombic) copper sites from UV-VIS spectroscopy. Computational modelling using an approximately 6 Å protein environment around the Cu site for both the oxidized and reduced forms showed that a small scale inner sphere rearrangement can account for the co-existence of two different redox active sites for a mononuclear cupredoxin independently from the employed density functionals. 1. Introduction Type 1 copper proteins function as electron carriers in many biological electron-transfer systems.1 Therefore, the electronic structure of the copper site has been studied extensively to elucidate the correlation between the structure, unique spectroscopic properties, and the Cu2+/Cu+ reduction potential.2-7 Electron paramagnetic resonance (EPR) spectroscopic features of the oxidized metalloprotein with unusual small hyperfine coupling (A||) value and the unique electronic absorption spectra from ultraviolet-visible absorption spectroscopy (UV-VIS) were attributed to trigonally (axial) or tetragonally (rhombic) distorted tetrahedral Cu coordination environments. The two different coordination geometries eliminate the Jahn-Teller distortion force for a four-coordinate d9 metal site. These axial and rhombic sites correspond to optically different blue and green Cu proteins, respectively. In addition to the Jahn-Teller effect, the geometric distortion can be rationalized by the entatic state or rack-induced bonding from the protein environment.3, 4 To date, the distortion model describing the presence of axial or rhombic sites invoked a single copper site.3 However, the possibility for the simultaneous presence of different copper sites in the same protein environment has already been suggested by early resonance Raman measurements.8 Furthermore, a flat potential surface along the Cu-S(Met) and Cu-S(Cys) distortion coordinates was demonstrated by computational modelling. Density functional theory (DFT) results show that large-scale structural distortions can take place involving the Cu site at negligible energetic costs.3, 9, 10 Importantly, the presence of a dual or a single Cu site as a stationary structure was dependent on the employed level of theory. Pure density functionals with the Generalized Gradient Approximation (GGA) for computational models containing the inner sphere environment9 localized two minima on the shallow potential energy surface. These correspond to axial (blue) and rhombic (green) Cu2+ sites. However, hybrid density functional studies with more complete basis sets consistently resulted in a single Cu site model.3, 11 In addition to the employed functional, it has been shown that the completeness of the computational model can be equally critical as much as the level of theory due to the necessary treatment of the network of weak interactions that involve the metal site and its inner-sphere protein environment.12-14 Pseudoazurin from Achromobacter cycloclastes (AcPAz) is an electron donor to nitrite reductase and nitrous oxide reductase in the denitrification process.15-17 In a previous study,18 the crystal structure of AcPAz was refined to 1.35 Å resolution (1BQK) with a single Cu site. The dual Cu positions could not be resolved from the electron density map. However, previous comprehensive spectroscopic observations indicated the simultaneous presence of two different Cu2+ sites.2, 19, 20 Improvement in crystallization conditions, thus in crystal quality, and developments of synchrotron beamlines and software resulted in identifying two positions for the Cu site in AcPAz at the resolution of 1.10 Å. perpendicular basal L-Cu-L’ angles correspond to the rhombic Cu site in AcNiR.34 The axial site in position 1 can be clearly identified by an approximately 0.5 Å longer Cu-S(Met86) distance in comparison to the rhombic site in position 2 (3.10 Å vs. 2.60 Å, respectively). Interestingly, the Cu-S(Cys78) distances in AcPAz for both Cu positions were found to be identical (2.21 Å). Generally, the Cu-S(Cys) distance in the axial site is expected to be shorter than the rhombic site from the higher Cu-S(Cys) frequency in resonance Raman spectra,19 and the increased Cu-S bond covalency from S K-edge XAS.21 Highly similar bond lengths for the oxidized axial and rhombic Cu sites in AcPAz were also observed by Cu K-edge extended X-ray absorption fine structure (EXAFS) analysis.21 The inconsistencies between the resonance Raman results and the XRD/XAS analyses can be resolved by considering a balance between covalent and ionic Cu-ligand interactions in determining the bond strength. Ionic interactions at short distances are of comparable strength to covalent interactions with good orbital overlap.21 The shorter Cu-N(His81) bond can be rationalized by the longer Cu-S(Met86) distance in the axial site in comparison to the rhombic site. The Cu-N bond lengths for the axial (1.86-2.03 Å) and rhombic (1.96-2.21 Å) sites of AcPAz show a significantly larger range than the corresponding distances in PnPc (1.94-1.99 Å) and AcNiR (2.03-2.04 Å). The Cu positions in PnPc and AcNiR were modelled by a single position despite the indication for the simultaneous presence of axial and rhombic sites by J-band EPR for plastocyanin20 and resonance Raman for nitrite reductase.8 The reported Cu-L distances in PnPc and AcNiR may well be considered as the population weighted average of shorter and longer Cu-N(His) distances in the axial and the rhombic sites, respectively. The EXAFS analyses of the wild type and several variants of AcPAz could not differentiate between the two Cu-N(His) distances either,21 since they show an average value of 1.94-1.96 Å similarly to those in PnPc and AcNiR XRD structures. The comparison of the two Cu-N(His) distances among the four crystallographic structures in Table 2 reveals a notable relationship. The Cu-N(His40) distance of position 1 (axial) is longer than position 2 (rhombic) by 0.07 Å for AcPAz. However, PnPc as the reference for axial Cu site has a 0.10 Å shorter bond length than rhombic site of AcNiR. Furthermore, in rhombic AcPAz the short Cu-N(His40) bond correlates with the long Cu-N(His81) bond (0.17 Å deviation), and vice versa for the axial AcPAz (0.25 Å deviation). In PnPc and AcNiR, these deviations are however only less than 0.05 Å. Given the high resolution of the crystal structures in Table 2, the origin of the peculiar trend in Cu-N ligand distances may lie in the second sphere or perhaps further outer sphere protein environment effects. The L-Cu-L’ angles for the axial and rhombic sites also differ significantly from each other. Moreover, we carefully considered whether the presence of the two different Cu positions was due to disorder in the coordinating amino acid (His40, Cys78, His81, and Met86). This was observed by others during the refinement of Cu positions of Met98Gln variant of amicyanin that showed disorder of Cu and Cu-coordinating Gln98 residue.33 The refinement of multiple Cu sites in the amicyanin structure necessitates the evaluation of the structural differences between oxidized and reduced forms. As we have seen in the EXAFS study,21 during X-ray irradiation the samples can undergo radiation damage, particularly photoreduction. The Cu-ligand distances in Type 1 copper site is expected to elongate upon Cu reduction due to an increased electrostatic repulsion between metal and ligands in addition to the loss of covalent interactions between Cu 3d and ligand 2p/3p orbitals. Therefore, the simultaneous presence of Cu2+ and Cu+ ions may provide an explanation for the dual Cu positions alternative to the presence of both axial and rhombic oxidized Cu sites. In order to evaluate this possibility, we examined the structural effects of reduction on the Cu coordination geometry in Type 1 copper proteins with [N2S2] coordination. The considered crystal structures were obtained under similar pH conditions for the same Type 1 copper proteins.18, 35, 37-41 Cu-ligand distances for all structures are summarized in Table S1 of Supporting Information. Surprisingly, some of the structures (#1-4 and #11-13 in Table S1) show the shortening of Cu-ligand distances upon Cu reduction. The average elongation of Cu-S(Met) for all the Type 1 copper proteins was 0.05±0.10 Å between the reduced and oxidized sites, which is inconsistent with the observed difference of 0.50 Å for the two Cu positions of AcPAz. By focusing in on the AcPAz structures. the average elongation of Cu-S(Met) (#5-8 in Table S1) upon reduction (0.11±0.05 Å) was found to be still too small. The corresponding changes in the Cu-S(Cys) and Cu-N(His) distances for all entries in Table S1 (-0.01±0.08 Å and 0.05±0.11 Å) or for the AcPAz structures only (0.05±0.04 Å and 0.12±0.15 Å) also do not support the alternative explanation of a redox dependent presence of the two Cu positions. Thus, the rationale for the presence of partially reduced Cu site can be excluded on the basis of available crystal structures. 3.2. Theoretical calculations The flexibility of the inner sphere coordination environment was evaluated for accommodating the two Cu sites within the same protein matrix. A systematic analysis was carried out for fully and partially optimized inner sphere coordination environments around the Cu site in the oxidized and reduced states using the given atomic resolution crystal structure of AcPAz. The full structural optimizations employing the saturated basis set for all atoms, pure GGA (BP86) and hybrid GGA (B3LYP and B38HFP86) functionals converge to a single Cu site structure with Cu-S(Met) distances of 2.53, 2.63, and 2.61 Å for the oxidized, and 2.58, 2.99, and 2.72 Å for the reduced forms (Figure 3). These results suggest that considering inner coordination sphere interactions alone, there is a single metal site geometry for the Type 1 copper proteins in agreement with the prediction from the previously defined coupled distortion coordinate model.3 The above structural optimization results show a notable repulsive nature of the popular B3LYP potential, where basis set saturation results in elongated metal-ligand distances.42 and reactivity19, 43-47, can provide comparable energetic strain to the Jahn-Teller distortion that moves the Cu site away from its lowest energy rhombic structure and trap it in a higher energy axial structure. The structural basis of a single minimum on a coupled geometric distortion coordinate between the apical Cu-S(Met) and the basal Cu-S(Cys) distances was previously developed using low resolution crystal structures (1.90-2.70 Å for NiR,48 1.33 Å for Pc,49 1.80 Å for cucumber basic protein50). Now with the availability of atomic resolution crystal structures and careful consideration of the electron density around the Cu site, the simultaneous presence of axial and rhombic sites can be realized within the same protein crystal. This confirmation is in agreement with the interpretation of a broad range of spectroscopic results and computational analyses. 4. Conclusions The structure of AcPAz at 1.10 Å resolution demonstrated the co-existence of axial and rhombic Cu sites in the same crystal. The occupation of Cu positions in a Type 1 site was found to be consistent with their population from independent EPR spectroscopic measurements. It is important to highlight that the independent XRD, EPR,2, 20 and XAS21 measurements were performed at approximately same temperatures. In our past experience, the AcPAz does not show thermochromic behaviour.2, 20 These results strongly suggest the possibility for the presence of dual Cu geometry in Type 1 copper proteins. The presence of two Cu sites was already proposed earlier on the basis of spectroscopic studies of AcPAz,2, 19, 20 AcNiR,8 and Pc20 metalloproteins. The characteristically elongated electron density maps are commonly found in other blue and green copper proteins. The structural and spectroscopic observations suggest that the dual Cu positions can be a common feature for all Type 1 copper proteins. The chemical functional relevance of the dual Cu sites may include versatility through allosteric interactions in electron-transfer processes at reduction potential that is adjustable on-demand. Furthermore, the presence of dual Cu sites may contribute to accommodating various redox partners as required in nitrite reductase versus nitrous oxide reductase. Kinetic experiments aimed at biological functional relevance are currently underway. The origin of the presence of two Cu positions cannot be correlated with a mixture of reduced and oxidized forms as described for amicyanine.33 It is more likely that protein dynamics is responsible for the dual Cu sites through a fascinating network of weak interactions. This is in contrast to previous models, where the Cu site moves its ligand environment due to Jahn-Teller distortion forces. Acknowledgements We are grateful to Professor Yasuhito Shomura of Ibaraki University for his insightful discussions. This work was supported by Grant-in-Aid for Scientific Research from JSPS No. 22550145 (TK) and the Sekisho Scholar Award (AT&TY). This work was also partly supported by the Reimei Kenkyu Award from Japan Atomic Energy Agency, JAEA (MU). The MTA-ELTE Chemical Structure & Function “Momentum” Laboratory (ID 96122) is supported by the Hungarian Academy of Sciences, Budapest, Hungary (Contract No. LP2015- 10/2015). Portion of this research was conducted at Photon Factory (PF) BL NW12A as part of proposals 2009G578, 2011G519, and 2013G504. Notes and references 1 C. Dennison, Cood. Chem. Rev., 2005, 249, 3025-3054. 2 R. F. Abdelhamid, Y. Obara, Y. Uchida, T. Kohzuma, D. M. Dooley, D. E. Brown and H. Hori, J. Biol. Inorg. Chem., 2007, 12, 165-173. 3 E. I. Solomon, R. K. Szilagyi, S. D. George and L. Basumallick, Chem. Rev., 2004, 104, 419-458. 4 L. B. LaCroix, S. E. Shadle, Y. N. Wang, B. A. Averill, B. Hedman, K. O. Hodgson and E. I. Solomon, J. Am. Chem. Soc., 1996, 118, 7755-7768. 5 S. DeBeer, D. W. Randall, A. M. Nersissian, J. S. Valentine, B. Hedman, K. O. Hodgson and E. I. Solomon, J. Phys. Chem. 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Biol., 1996, 262, 686-705. 203x101mm (300 x 300 DPI) Simultaneous presence of axial (blue) and rhombic (green) Cu-site in pseudoazurin is described from experiments and computational modelling.