Crystal Structure Determinations of Oxidized and Reduced Pseudoazurins from Achromobacter cycloclastes
CONCERTED MOVEMENT OF COPPER SITE IN REDOX FORMS WITH THE REARRANGEMENT OF HYDROGEN BOND AT A REMOTE HISTIDINE*

Tsuyoshi Inoue, Nobuya Nishio, Shinnichiro SuzukiDagger , Kunishige KataokaDagger , Takamitsu Kohzuma§, and Yasushi Kai

From the Department of Materials Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan, the Dagger  Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan, and the § Department of Chemistry, Faculty of Science, Ibaraki University, Mito, Ibaraki 310-8512, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The crystal structures of oxidized and reduced pseudoazurins from a denitrifying bacterium, Achromobacter cycloclastes IAM1013, have been determined at 1.35- and 1.6-Å resolutions, respectively. The copper site in the oxidized state exhibits a distorted tetrahedral structure like those of other pseudoazurins. However, not only a small change of the copper geometry, but concerted peptide bond flips are identified. The imidazole ring of remote His6 has a hydrogen bonding distance of 2.73 Å between N-delta 1(His6) and O-gamma 1(Thr36) in the oxidized protein. When the protein is reduced at pH 6.0, the imidazole ring rotates by 30.3° and moves 1.00 Å away from the position of the oxidized state. A new hydrogen bond between N-epsilon 2(His6) and O-epsilon 1(Glu4) is formed with a distance of 3.03 Å, while the hydrogen bond between N-delta 1(His6)-O-gamma 1(Thr36) is maintained with an interatomic distance of 2.81 Å. A concomitant peptide bond flip of main chain between Ile34 and Thr36 occurs.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pseudoazurin (Mr ~ 14,000) is a type 1 blue copper protein (cupredoxin) found in denitrifying bacteria and methylotrophs. While in methylotrophs pseudoazurins were produced as electron transfer substituted to another blue copper protein named amicyanin at high copper concentration. The ternary complex among methylamine dehydrogenase, amicyanin, and cytochrome c551 shows that amicyanin accepts an electron from methylamine dehydrogenase (1). On the other hand, in denitrifying bacteria pseudoazurins donate an electron for the reduction of NO2- to NO by nitrite reductase under anaerobic conditions (2-5). Pseudoazurins from four sources, Achromobacter cycloclastes IAM1013 (2, 6, 7), Methylobacterium extorquens AM1 (8), Alcaligenes faecalis S-6 (3, 9), and Thiosphaera pantotropha (10) have been characterized to date. From their amino acid sequences, it is known that A. cycloclastes pseudoazurin has an extra C-terminal residue giving 124 amino acids in total (11). The greatest degree of similarity among pseudoazurins is 65% conservation of amino acid residues between A. cycloclastes and A. faecalis.

The crystal structure analyses of pseudoazurins from A. faecalis (11-14), T. pantotrophs (15), and M. extorquens (16) revealed that the overall topology of pseudoazurin consists of an eight-stranded beta -barrel which resembles the structures of other cupredoxins like plastocyanin and azurin. Differences occur between these proteins regarding the amount and location of helical structure. Pseudoazurin possess two extra alpha -helices at the C terminus, whereas azurin has an alpha -helical flap in the middle of the sequence and plastocyanin generally has a small (1 turn) helix (17). The copper atom is located below the protein surface at a depth of 5-10 Å, with two histidines (imidazole N), a cysteine (thiolate RS-), and methionine (thioether S) as ligands (18). Nine of 13 lysine residues surrounded the hydrophobic face of the molecule through which the histidine ligand protrudes slightly. The effect of lysine residues for the electron transportation from pseudoazurin to nitrite reductase was well investigated by using mutant pseudoazurin from A. faecalis S-6 (19). The substitution of these basic lysine residues by amino acids with neutral/acidic side chains decreases the affinity of pseudoazurin for nitrite reductase, which suggests that pseudoazurin may interact with nitrite reductase through its hydrophobic patch. Moreover, the structure of pseudoazurin from T. pantotrophs revealed that the proposed docking motifs based on the positive hydrophobic surface patch for cytochrome cd1 nitrite reductase (15).

Small inorganic complexes such as [Fe(CN)6]3- and [Co(phen)3]3+ (phen = 1,10-phenanthroline) have been used as redox probes to identify potential binding sites on the surface of cupredoxins for electron-transfer partners (20). Recent kinetic and theoretical studies on the blue copper protein plastocyanin have indicated the presence of two distinct electron-transfer sites: (i) the adjacent hydrophobic patch ~6 Å from the copper through which one of the histidine ligands protrudes, and (ii) the remote site involving and acidic patch region ~15 Å from the copper, with acidic residues on either side of the exposed Tyr83 (Tyr83 is next to the copper ligand Cys84). Kinetics studies with small inorganic complexes have been carried out on A. cycloclastes pseudoazurin. The rate constant for the oxidation of the reduced molecule with [Fe(CN)6]3- is 103-fold bigger than that for the [Co(phen)3]3+ oxidation (21), indicating a clear preference of the protein for anionic species. Moreover, the self-exchange rate constant for reduced molecule (about 3 × 103 M-1 s-1) is much smaller than those of most other cupredoxins and is quite similar to that found in higher plant plastocyanins (22). A. cycloclastes nitrite reductase, which possesses many acidic amino acid residues, accepts an electron from a reduced pseudoazurin with a second-order rate constant of 7-9 × 105 M-1 s-1 (5, 23). These findings are all probably due to the effect of a number of conserved lysine residues surround the hydrophobic patch of pseudoazurin.

The effect of pH on the redox reactivity of A. cycloclastes pseudoazurin is well documented (21, 22, 45). The reduced protein has been shown to participate in an active site protonation/deprotonation equilibrium at His81, which gives an acid dissociation pKa of 5.2 from an NMR titration (21) and 4.6 from kinetic studies with inorganic complexes as oxidants (45). The effect of protonation/deprotonation of uncoordinated His6 residue located near to the active site shows a pKa values of 7.2 (reduced form) and 6.5 (oxidized form) from NMR titration and 7.3 from kinetic studies of the oxidation of the reduced pseudoazurin with small complexes (21).

The structure of reduced pseudoazurin from A. faecalis at pH 4.4 has been solved and demonstrates that the ligand His81 rotates away from the metal due to protonation of the N-delta 1 atom (14). Small changes were observed in the copper vicinity and on the protein surface at pH 7.8 upon reduction (14), while distinct conformational changes occurred in response to reduction at pH 7.0: the copper position shifted, Met7 and Pro35 moved, and the position of solvent molecules changed (24). Preliminary crystallographic studies of A. cycloclastes pseudoazurin were reported by Turley et al. (25), but the crystals obtained were too thin for x-ray diffraction data to be obtained. We have previously reported the crystallization and preliminary x-ray studies on oxidized pseudoazurin from A. cycloclastes at pH 6.0 (26). In this work single crystals of oxidized and reduced pseudoazurin with high resolutional diffraction spots were obtained at pH 6.0. During the last stage of structure refinement, the amino acid sequence was partially corrected (27).1 We describe here the redox-induced conformational changes at the copper site, and the rearrangement of the hydrogen bonding pattern of the uncoordinated His6, which are quite similar, but not identical, to the pattern of changes found in A. faecalis pseudoazurin reduced at pH 7.0 (24). The peptide flip induced by protonation at His6 is pH-induced conformational transition of His35 in azurin from Pseudomonas aeruginosa (28).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Data Collection and Processing-- The crystallization (0.1 M potassium phosphate buffer (pH 6.0)) and structural analysis of oxidized pseudoazurin from A. cycloclastes were performed as described previously (26). The data with high resolution was collected with synchrotron radiation at the BL6A2 station of 2.5 GeV energy produced by the storage ring in the Photon Factory, the National Laboratory for High Energy Physics (KEK), Tsukuba, Japan. Diffraction patterns were recorded on a Fuji Imaging Plate (200 × 400 mm, Fuji Photo Film) (29) using Sakabe's Weissenberg camera for macromolecules (30) with an aperture collimator of 0.1-mm diameter and a cylindrical cassette of 286.5-mm radius filled with helium gas. The intensity data were processed using DENZO and scaled with the program SCALEPACK (31). Among 222,685 accepted observations up to 1.35-Å resolution, 21,303 independent reflections were obtained, the completeness of which was 88.8% with an Rmerge value of 8.5%. The reduced crystals of pseudoazurin were obtained by soaking the oxidized crystals with the crystallization solution (0.1 M potassium phosphate, pH 6.0) containing 10 mM sodium L-(+)ascorbate. After 10 min the blue crystals became colorless. For more than 1 month the crystals remained colorless in the glass capillary with the mother liquor containing 10 mM sodium L-(+)ascorbate. The x-ray diffraction data up to 1.6-Å resolution were obtained using the imaging plate detector operated in the Rigaku RAXIS-IIc system. Among 58,870 accepted observations up to 1.60-Å resolution, 13,287 independent reflections were obtained, the completeness of which was 90.4% with a Rmerge values of 4.0%.

Structure Analysis and Refinement-- Structure analysis has been carried out by the molecular replacement method using the MERLOT program package (32). Calculations using the molecular structure of pseudoazurin from A. faecalis as a starting model were performed. The highest rotation peak was found at Eulerian angles of alpha  = 115.0°, beta  = 60.0°, and gamma  = 305.0° for 13-3.2-Å resolution data. The translation parameters were calculated in the RVAMAP function with MERLOT, and the highest peak (x = 0.45, y = 0.10, and z = 0.15 in fraction of unit cell) gave the minimum R-factor of 64.9% for 15-3.88-Å resolution data. After several cycles of the RMINIM function, the rotation parameters converged to alpha  = 115.40°, beta  = 62.23°, and gamma  = 303.99°, and the translation parameters to × = 0.4537, y = 0.1330, and z = 0.1387. The R-factor calculated for the model structure was 47.5% for 15.0-3.88-Å data. Refinements for both oxidized and reduced structures have been carried out by using PROLSQ: (33), X-PLOR version 3.0 (34), and TURBO-FRODO (35). The first step of the refinement for both forms was performed using X-PLOR. A simulated annealing calculation at 3000 K, further several steps of positional refinement were performed by X-PLOR using the stepwise increased data. Because of the ambiguity for the energy constraint parameters of metal atom used in the molecular dynamics refinement, somewhat unreliable copper geometries were obtained. In order to obtain exact copper geometry no restraint was imposed on the copper coordination through the final stage of refinement by REFMAC. Every stage includes model improvement followed by several cycles of refinement. Model improvements were carried out based on the Fourier maps calculated with the coefficients of (2Fo - Fc)exp(2pi ialpha calc) and (Fo - Fc)exp(2pi ialpha calc). The final structure model, further rebuilding, and cycles of refinement for the water structure of oxidized molecule finally decreased the R-factor to 17.6% for the data between 8.0- and 1.35-Å resolution with quite reasonable stereochemistry. Rfree (37) was 18.9% for 5% of total data within the same resolution range. On the other hand, the final R- and Rfree-factors for the reduced structure by using the data between 8.0- and 1.6-Å resolution were 17.3 and 21.1%. The results of data collection and refinement are summarized in Table I.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Quality of the Final Model-- The final model of oxidized pseudoazurin from A. cycloclastes is made up of one monomer in the asymmetric unit with 124 amino acids, 121 water molecules, and single copper ion. The model remained close to standard geometry throughout refinement. The final model of reduced structure includes 96 water molecules. The mean positional errors of the atoms estimated by Luzzati plots are 0.137 Å for the oxidized protein and 0.153 Å for reduced form, respectively (38). For well defined parts of the structure, especially the beta -strands, the internal side chains and the region around the metal site, the errors are likely to be lower. The quality of the final model is summarized in Table I. The program PROCHECK (39) was used to analyze conformational variations from defined norms. A Ramachandran plot (40) shows that all non-glycine residues have dihedral angles falling in (or near to) energetically preferred regions.

                              
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Table I
Data collection and final refinement statistics for the oxidized and reduced pseudoazurins from Ach. cycloclastes

Overall Structure-- A ribbon drawing of A. cycloclastes pseudoazurin is presented in Fig. 1. The approximately spherical pseudoazurin molecule has overall dimensions of 38 × 38 × 27 Å. The molecule possesses eight beta -strands, forming two beta -sheets, and two C-terminal alpha -helices. beta -Sheet I consists of four beta -strands: S1, residues 2-8; S2a, 17-19; S3, 30-34; S6, 64-67, and beta -sheet II contains five beta -strands: S2b, residues 22-25; S4, 42-44; S5, 56-58; S7, 72-77; S8, 87-92. These structural features are very similar to those of the other pseudoazurins (from A. faecalis, M. extorquens, and T. pantotropha) (11-16). The number of identical residues are 81/124 (65%) between pseudoazurins from A. cycloclastes and A. faecalis, 64/123 (52%) between pseudoazurins from A. cycloclastes and M. extorquens, and 54/123 (44%) between pseudoazurins from M. extorquens and A. faecalis. The three structures are so analogous, except at the C-terminal alpha -helices, that averaged r.m.s.2 deviations of the backbone structures among three pseudoazurins are less than 0.7 Å (0.66 Å, 0.55 Å, and 0.69 Å, respectively).


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Fig. 1.   Ribbon presentation of the oxidized pseudoazurin from A. cycloclastes viewed from the side of the molecule (a) and from above the copper site (b). The copper ion is shown with a sphere in red at the top of the model. Four ligands, His40, Cys78, His81, and Met86, are represented by ball-and-stick model. The uncoordinated His6 and basic residues (Arg and nine Lys) surrounding the hydrophobic surface are also shown with ball-and-stick. The figure is drawn by program MOLSCRIPT (43) and RASTER3D (44).

Conformational Changes in the Copper Vicinity-- A dramatic change at the copper center, from tetrahedral to trigonal, was observed at pH 4.4 in A. faecalis pseudoazurin upon reduction (14) as well as in poplar plastocyanin at pH 3.8 (41). However, only small changes between oxidized and reduced pseudoazurins from A. faecalis were observed at pH 7.8 (14), while shifts around the copper ion were observed upon reduction at pH 7.0 (24). For A. cycloclastes pseudoazurin, the r.m.s. deviation of backbone structures between the two oxidation states of the protein is 0.14 Å (Fig. 2a). The conformational differences of the copper geometries in the oxidized and reduced forms are shown in Fig. 2b. The copper-His81 distance is significantly lengthened by the reduction of the copper center (0.19 Å) in contrast to the small lengthening of other bonds (copper-His40 (0.09 Å), copper-Cys78 (0.06 Å), and copper-Met86 (0.14 Å)). The cuprous center also has a distorted tetrahedral geometry. The bond lengths and angles at the copper centers are summarized in Table II.


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Fig. 2.   Superimposition of the oxidized (thin lines) and reduced (thick lines) pseudoazurins from A. cycloclastes for whole structures (a) and the copper sites (b). A quite small r.m.s. deviation of 0.14 Å is calculated for whole structures. However, the copper site and the uncoordinated His6 are significantly moved on reduction at pH 6.0.

                              
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Table II
Metal coordinations of pseudoazurins

Other Redox Linked Conformational Changes-- Fig. 3, a and b, show that both models are well fitted to the electron density maps. The structure comparison between the oxidized form and the reduced form is performed by least square method with all backbone atoms. Despite the quite small r.m.s. deviation of 0.14 Å for the backbone, some significant differences are found. The striking finding is the rearrangement of hydrogen bonding interactions of the non-coordinated His6 and the peptide bond flip around Pro35 as shown in Fig. 3c. The N-delta 1(His6) forms a hydrogen bond to O-gamma 1(Thr36) with a distance of 2.73 Å in the oxidized pseudoazurin. Both the O-gamma 1(Thr36) and the N-delta 1(His6) atoms possess relatively low temperature factors of 14.4 and 16.1 Å2, respectively. However, the imidazole ring of His6 moves through a distance of 1.00 Å and the dihedral angle between the hydrogen bond and the imidazole ring changes by 30.3° on reduction at pH 6.0. Accompanying this change the N-epsilon 2 of His6 to O-epsilon 2 of Glu4 distance decreases from 3.47 Å to 3.07 Å forming a new hydrogen bond (Fig. 3c). Apparently the hydrogen bonding between His6 and Thr36 is weakened. The rearrangement of hydrogen bond of His6 leads to a concomitant Ile34-Thr36 main chain peptide bond flip. The main chain conformational angles are also changed dramatically, particularly around Pro35 (Table III). The Calpha and Cgamma atoms of Pro35 have moved by 0.93 and 2.16 Å, respectively.




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Fig. 3.   The oxidized (a) and reduced (b) structures of pseudoazurin from A. cycloclastes with omit maps. Structural comparison around copper site and His6 between the oxidized (line in blue) and reduced (line in magenta) forms (c). The copper coordination structure is changed from blue line to yellow line on reduction. Concerted movements around His6 at the distance of 12 Å from the copper ion are shown in ball-and-stick models colored in blue (oxidized form) and red (reduced form). The dihedral angle between the hydrogen bond of N-delta 1(His6)-O-gamma 1(Thr36) and the imidazole ring was changed from 0.24° to -30.1° upon reduction. Namely, the imidazole ring moved through a distance of 1.00 Å and rotated by 30.3° against the relative position of O-gamma 1(Thr36). On the contrary, the distance of O-epsilon 2(Glu4)-N-epsilon 2(His6) is changed from 3.47 Å to 3.07 Å and a new hydrogen bonding is formed. C-alpha (Pro35) and C-gamma (Pro35) moved 0.93 Å and 2.16 Å, respectively, and the dihedral angle of Calpha -Cbeta and Cgamma -Cdelta in Pro35 was changed by 30.9°. The water molecule (green color), which locates in the oxidized state with a temperature factor of 13.9 Å2 at distances of 2.92 and 2.83 Å from N(His40) and O(Asp37), respectively, was lost after reduction. This figure is drawn by program PROTEUS (System Co., Ltd., Japan).

                              
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Table III
Change of main chain conformational angles

Loss of Water Molecules Adjacent to Gly39-- In azurin the backbone carbonyl oxygen of Gly45 provides a second weakly interacting axial ligand at a distance of 3.1 Å from Cu2+, resulting in a distorted trigonal bypyramidal coordination geometry. In plastocyanins and pseudoazurins the corresponding distance is longer than that in azurin, and the active site geometry is distorted tetrahedral. The O(Gly39) of A. cycloclastes pseudoazurin, which corresponds to O(Gly45) in azurins, locates at a distance of 3.94 Å from the copper ion in the oxidized protein, and a water molecule with a temperature factor of 13.9 Å2 is found at distances of 2.92, 2.83, and 3.04 Å from N(His40), O(Asp37), and O(Asn61), respectively (Fig. 3c). Because the O(Gly39) is adjacent atom of N(His40), the water molecule forms a long range hydrogen-bonding network ranging from O(Gly39) to O(Asn61). However, upon reduction the distance between O(Gly39) and the copper ion is lengthened to 4.04 Å, which shows the extension of ionic radius of the copper center. Moreover, the water molecule is lost providing space for the peptide bond flip of Pro35. The conformations of the main chain around Gly39 is changed as shown in Table III. The extended ionic radius of copper and the reduction of the copper charge may influence the water molecule, which may facilitate the peptide bond flip and the rearrangement of the hydrogen bonding of His6.

Structural Comparison between Pseudoazurins from A. cycloclastes and A. faecalis-- The oxidized and reduced pseudoazurins from A. cycloclastes are superimposed on those from A. faecalis, respectively, with r.m.s. deviations for backbone atoms of 0.64 and 0.62 Å. The superimposition reveals the remarkable structural differences at the imidazole ring of His6 (Fig. 4). The angles between the imidazole rings of His6 in A. cycloclastes and A. faecalis pseudoazurins are different by more than 70°. The imidazole ring rotates to the perpendicular position relative to that in A. faecalis, which enables it to form the strong hydrogen bond to Thr36 in the oxidized pseudoazurin from A. cycloclastes. The residue Glu4 is conserved in both pseudoazurin but the 36th amino acid residue is Val instead of Thr in A. faecalis pseudoazurin. Only one hydrogen bond is therefore possible between His6 and Glu4. The N-epsilon 2(His6)-O-epsilon 1(Glu4) distances are 2.76 Å (oxidized form) and 2.96 Å (reduced form) at pH 7.8 (14) in A. faecalis pseudoazurin, while the distances are 2.81 Å (oxidized form) and 2.71 Å (reduced form) at pH 7.0 (24). The rearrangement of hydrogen bond of His6 was not observed in pseudoazurin from A. faecalis S-6. On the other hand, Thr36 fixes the position of His6 in the direction of O-gamma 1(Thr36) atom and the N-epsilon 2(His6) is directed toward the side chain of Glu4 in the oxidized form of pseudoazurin from A. cycloclastes. Thus, whether threonine or valine at residue 36 is the clue to the appearance of redox-induced rearrangement of hydrogen bonding around His6.


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Fig. 4.   The superimposed structures of oxidized and reduced pseudoazurins from A. cycloclastes (thin lines) and those from A. faecalis (thick lines). The aromatic ring of His6 rotates by 74° to the perpendicular position capable of forming the hydrogen bond to Thr36 in the oxidized pseudoazurin from A. cycloclastes. The replacement of Val36 by Thr36 fixes the protonation site inward the protein surface by the hydrogen bond, which is proved to be the key factor of the concerted movement found in A. cycloclastes pseudoazurin.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

According to the detailed structural comparison between the oxidized and reduced pseudoazurin from A. cycloclastes, the difference in copper-His81 distance is bigger between two forms than other copper-ligand distances. The expansion of the ionic radius of the copper, 0.96 Å for Cu1+ and 0.69 Å for Cu2+, and the reduction of copper charge upon reduction results in the loss of a water molecule situated close to the active site. For this reason the peptide bond flip at Pro35 may occur more easily. The loss of a similar water molecule was found in A. faecalis pseudoazurin at both pH 7.8 and 7.0, while the peptide bond flip was found at only pH 7.0. Because the pKa values of the uncoordinated His6 residue are 7.2 (reduce form) and 6.5 (oxidized form) (21), the protonation at His6 may be the cause of the peptide bond flip in Al. faecalis pseudoazurin. A similar peptide bond flip was observed in oxidized azurin from P. aeruginosa upon changing the pH from 9.0 to 5.5 (28). In this case the rearrangement of the hydrogen bonding pattern resulted from the protonation/deprotonation of His35 and induced the peptide bond flip. The vicinity of the copper site was not significantly affected by this conformational change. However, in this study all processes have been performed at pH 6.0, both forms have the protonated His6. The protonation may not be the direct trigger of the peptide bond flip in this study. Actually, the strong hydrogen bond between His6 and Thr36 is weakened, and a new hydrogen bond between His6 and Glu4 is formed on reduction. The N-epsilon 2 atom of His6 should carry a proton on reduction because it can only be a proton donor to Glu4. On the other hand, N-delta 1 may also be a protonated, but it depends if O-gamma 1(Thr36) acts as acceptor. Despite the small space between O-gamma 1(Thr36) and Ndelta 1(His6), it is available to add a proton on the N-delta 1(His6) atom by a free rotation around Cbeta -Ogamma 1 in Thr36 of the oxidized protein. Both the protonated nitrogen atom of His6 is consistent with the peptide bond flip. The geometrical changes at the copper center have been accompanied by both the peptide bond flip and the loss of water molecule. Because the metal site consists of the copper ion and three strong ligand atoms (two histidines and a cysteine), the water molecule may exist as a hydronium ion to compensate the excess minus charge at the copper site in the oxidized pseudoazurin. The loss of water molecule may occur for the reduction of copper plus charge. This may be the direct trigger of the concerted movement around His6. However, it may be said that both a loss of water molecule and a protonation at the remote histidine may need for the peptide bond flip, because the peptide bond flip was found only at pH 7.0 in the pseudoazurin from A. faecalis.

The rearrangement of hydrogen bonding interactions of the uncoordinated His6 are observed in A. cycloclastes pseudoazurin having Thr36, but not in A. faecalis pseudoazurin with Val36. An important difference between these pseudoazurins is that Thr36 fixes the position of the imidazole ring of His6 by forming the strong hydrogen bond to its N-delta 1 atom. On the other hand, Val36 could not fix the nitrogen atom of His6 and the hydrogen bond between N-epsilon 2(His6) and O-epsilon 1(Glu4) was already formed in the oxidized state. This is why a small peptide bond flip was observed in A. faecalis pseudoazurin at pH 7.0 (24).

The protonated pseudoazurin at the His6 position indicated a relatively higher redox potential (42). The protonation at His6 is important to reduction of the protein, and then, the peptide bond flip occurs in the concomitant region from Ile34 to Thr36 upon reduction. Since the His6 residue is not so far from the copper site (12 Å), this fact might be associated with the the electron transfer mechanism.

    ACKNOWLEDGEMENTS

We are grateful for helpful and stimulating discussions of Dr. Chris Dennison, University College Dublin. We are also grateful to Professor N. Sakabe, Dr. N. Watanabe, Dr. Suzuki, and Dr. Igarashi for support in data collection at KEK, Japan.

    FOOTNOTES

* This work was supported in part by Grant-in-Aids 09261223, 03241215, 04225216, 05209216, and 08249221 for Scientific Research on Priority Areas, Grants-in-Aid 11780495, 09780632, and 07780572, for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan, and the Tsukuba Advanced Research Alliance (TARA) Sakabe project.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1BQK and 1BQR) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

To whom correspondence should be addressed. Tel.: 81-6-6879-7408; Fax: 81-6-6879-7409; E-mail: kai{at}chem.eng.osaka-u.ac.jp.

1 R. P. Ambler and M. Daniel, private communication.

    ABBREVIATIONS

The abbreviation used is: r.m.s., root mean square.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Chen, L., Durley, R. C. E., Mathews, F. S., and Davidson, V. L. (1994) Science 264, 86-90[Medline] [Order article via Infotrieve]
  2. Liu, M. Y., Liu, M. C., Payne, W. J., and LeGall, J. (1986) J. Bacteriol. 166, 604-608[Medline] [Order article via Infotrieve]
  3. Kakutani, T., Watanabe, H., Arima, K., and Beppu, T. (1981) J. Biochem. (Tokyo) 89, 463-472[Abstract]
  4. Moir, J. W. B., Baratta, D., Richardson, D. J., and Ferguson, S. J. (1993) Eur. J. Biochem. 212, 377-385[Abstract]
  5. Kohzuma, T., Takase, S., Shidara, S. & Suzuki, S. (1993) Chem. Lett. 149-152
  6. Iwasaki, H., and Matsubara, T. (1973) J. Biochem. (Tokyo) 73, 659-661[Medline] [Order article via Infotrieve]
  7. Ambler, R. P. (1997) in The Evolution of Metalloenzymes, Metalloproteins and Related Materials (Leigh, G. J., ed), pp. 100-118, Symposium Press, London
  8. Ambler, R. P., and Tobari, J. (1985) Biochem. J. 232, 451-457[Medline] [Order article via Infotrieve]
  9. Hormel, S., Adman, E. T., Walsh, K. A., Beppu, T., and Titani, K. (1986) FEBS Lett. 197, 301-304[CrossRef][Medline] [Order article via Infotrieve]
  10. Chan, C., Willis, A. C., Robinson, C. V., Aplin, R. T., Radford, S. E., and Ferguson, S. J. (1995) Biochem. J. 308, 585-590[Medline] [Order article via Infotrieve]
  11. Petratos, K., Banner, D. W., Beppu, T., Wilson, K. S., and Tsernoglou, D. (1987) FEBS Lett. 218, 209-214[CrossRef][Medline] [Order article via Infotrieve]
  12. Petratos, K., Dauter, Z., and Wilson, K. S. (1988) Acta Cryst. Sect. B 44, 628-636[CrossRef][Medline] [Order article via Infotrieve]
  13. Adman, E. T., Turley, S., Bramson, R., Petratos, K., Banner, D., Tsernoglou, D., Beppu, T., and Watanabe, H. (1989) J. Biol. Chem. 264, 87-99[Abstract/Free Full Text]
  14. Vakoufari, E., Wilson, K. S., and Petratos, K. (1994) FEBS Lett. 347, 203-206[CrossRef][Medline] [Order article via Infotrieve]
  15. Williams, P. A., Fulop, V., Leung, Y.-C., Chan, C., Moir, J. W. B., Howlett, G., Ferguson, S. J., Radford, S. E., and Hadju, J. (1995) Nat. Struct. Biol. 2, 975-982[Medline] [Order article via Infotrieve]
  16. Inoue, T., Kai, Y., Harada, S., Kasai, N., Ohshiro, Y., Suzuki, S., Kohzuma, T., and Tobari, J. (1994) Acta Cryst. Sect. D 50, 317-328[CrossRef][Medline] [Order article via Infotrieve]
  17. Adman, E. T. (1985) in Topics in Molecular and Structural Biology, Metalloproteins (Harrison, P., ed), Vol. I, pp. 1-42, Macmillan Ltd., New York
  18. Adman, E. T. (1991) in Advances in Protein Chemistry Copper Protein Structures (Anfinsen, C. B., Edsall, J. T., Richards, F. M., and Eisenberg, D. S., eds), pp. 145-197, Academic Press, New York
  19. Kukimoto, M., Nishiyama, M., Ohnuki, T., Turley, S., Adman, E. T., Horinouchi, S., and Beppu, T. (1995) Protein Eng. 8, 153-158[Abstract]
  20. Sykes, A. G., Kyritsis, P., Nordling, M., and Young, S (1993) in Bioinorganic Chemistry of Copper: Electron Transfer Reactivity of Mutants of the Blue Copper Protein Plastocyanin (Karlin, K. D., and Tyeklar, Z., eds), Vol. 1, pp. 78-99, Chapman & Hall, New York
  21. Dennison, C., Kohzuma, T., McFarlane, W., Suzuki, S., and Sykes, A. G. (1994) Inorg. Chem. 33, 3299-3305
  22. Dennison, C., Kohzuma, T., McFarlane, W., Suzuki, S. & Sykes, A. G. (1994) J. Chem. Soc. Dalton Trans. 437-443
  23. Kashem, M. A., Dunford, H. B., Liu, M.-Y., Payne, W. J., and LeGall, J. (1987) Biochem. Biophys. Res. Commun. 145, 563-568[Medline] [Order article via Infotrieve]
  24. Libeu, C. A. P., Kukimoto, M., Nishiyama, M., Horinouchi, S., and Adman, E. T. (1997) Biochemistry 36, 13160-13179[CrossRef][Medline] [Order article via Infotrieve]
  25. Turley, S., Adman, E. T., Sieker, L. C., Lie, M.-Y., Payne, W. J., and LeGall, J. (1988) J. Mol. Biol. 200, 417-419[Medline] [Order article via Infotrieve]
  26. Inoue, T., Nishio, N., Kai, Y., Harada, S., Ohshiro, Y., Suzuki, S., Kohzuma, T., Shidara, S., and Iwasaki, H. (1993) J. Biochem. (Tokyo) 114, 761-762[Abstract]
  27. Chen, J.-Y., Chang, W.-C., Chang, T., Chang, W.-C., Lie, M.-Y., Payne, W. J., and LeGall, J. (1996) Biochem. Biophys. Res. Commun. 219, 423-428[CrossRef][Medline] [Order article via Infotrieve]
  28. Nar, H., Messerschmidt, A., Huber, R., Kamp, M., and Canters, G. W. (1991) J. Mol. Biol. 221, 765-772[CrossRef][Medline] [Order article via Infotrieve]
  29. Miyahara, J., Takahashi, K., Amemiya, Y., Kamiya, N., and Satow, Y. (1986) Nucl. Instrum. Methods Phys. Res. A 246, 572
  30. Sakabe, N. (1991) Nucl. Instrum. Methods Phys. Res. A 303, 448-463
  31. Otwinowsk, Z., and Minor, W. (1996) Methods Enzymol 276, 307-326
  32. Fitzgerald, P. M. (1988) J. Appl. Crystallogr. 21, 273-278[CrossRef]
  33. Hendrickson, W. A. (1985) Methods Enzymol. 114, 252-270
  34. Brünger, A. T., Kurian, J., and Karplus, M. (1987) Science 235, 458-460
  35. Jones, T. A. (1978) J. Appl. Cryst. 11, 268-272
  36. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Cryst. Sect. D 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
  37. Brünger, A. T. (1992) Nature 355, 472-474[CrossRef]
  38. Luzzati, V. (1952) Acta Crystallog. Sect. A 5, 802-810[CrossRef]
  39. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallog. 26, 283-291[CrossRef]
  40. Ramachandra, G. N., and Sasisekharan, V. (1968) Adv. Protein Chem. 23, 283-437[Medline] [Order article via Infotrieve]
  41. Guss, J. M., Harrowell, P. R., Murata, M., Norris, V. A., and Freeman, H. C. (1986) J. Mol. Biol. 192, 361-387[Medline] [Order article via Infotrieve]
  42. Kohzuma, T., Yamada, M., Deligeer, and Suzuki, S. (1997) J. Elect. Anal. Chem. 438, 49-53
  43. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
  44. Merrit, E. A., and Murphy, M. E. (1994) Acta Cryst. Sect. D 50, 869-873[CrossRef][Medline] [Order article via Infotrieve]
  45. Dennison, C., Kohzuma, T., McFarlane, W., Suzuki, S. & Sykes, A. G. (1994) J. Chem. Soc. Commun. 581-582


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