Novel Insight into the Copper-Ligand Geometry in the Crystal Structure of Ulva pertusa Plastocyanin at 1.6-Å Resolution
STRUCTURAL BASIS FOR REGULATION OF THE COPPER SITE BY RESIDUE 88*

Naoki ShibataDagger , Tsuyoshi Inoue, Chizuko Nagano, Nobuya Nishio, Takamitsu Kohzuma§, Kazuhiko Onodera§, Fuminori Yoshizaki, Yasutomo Sugimura, and Yasushi Kaiparallel

From the Department of Applied Chemistry, Faculty of Engineering, Osaka University, Suita, Osaka 565-0871, Japan, the § Department of Chemistry, Ibaraki University, Mito, Ibaraki 310-8512, Japan, and the  Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274-8510, Japan

    ABSTRACT
Top
Abstract
Introduction
References

The crystal structure of plastocyanin from a green alga, Ulva pertusa, has been determined at 1.6-Å resolution. At its copper site, U. pertusa plastocyanin has a distorted tetrahedral coordination geometry similar to other plastocyanins. In comparison with structures of plastocyanins reported formerly, a Cu(II)-Sdelta (Met92) bond distance (2.69 Å) is shorter by about 0.2 Å and a Cu(II)-Sgamma (Cys84) distance is longer by less than 0.1 Å in U. pertusa plastocyanin. These subtle but significant differences are caused by the structural change at a His-Met loop (His87-Met92) due to an absence of a O(Asp85)-Ogamma (Ser88) hydrogen bond which is found in Enteromorpha prolifera plastocyanin. In addition, poplar and Chlamydomonas reinhardtii plastocyanins with a glutamine at residue 88 have a weak cation-pi interaction with Tyr83. This interaction lengthens the Cu(II)-Sdelta (Met92) bond of poplar and C. reinhardtii plastocyanins by 0.14 and 0.20 Å, respectively. As a result of structural differences, U. pertusa plastocyanin has a less distorted geometry than the other plastocyanins. Thus, the cupric geometry is finely tuned by the interactions between residues 85 and 88 and between residues 83 and 88. This result implies that the copper site is more flexible than reported formerly and that the rack mechanism would be preferable to the entatic theory. The His-Met loop may regulate the electron transfer rate within the complex between plastocyanin and cytochrome f.

    INTRODUCTION
Top
Abstract
Introduction
References

Plastocyanin is a key metalloprotein in the electron transfer processes from photosystem II to photosystem I. Plastocyanin consists of a polypeptide of approximate 100 amino acid residues and a type 1 copper atom (also called the "blue copper site") coordinated by the side chains of four residues, two histidines, a cysteine, and a methionine. Plastocyanin has 2 electron transfer paths. One is the hydrophobic patch consisting of hydrophobic residues at the northern end of the molecule (1) and the electron transfer occurs through His87. Another site is called negative patch or acidic patch located at the east end of the molecule and it has Tyr83, called a remote site, as one of the electron paths to the copper atom.

Crystal structure analyses of oxidized plastocyanins from poplar (2-4), green algae Enteromorpha prolifera (5), and Chlamydomonas reinhardtii (6), have been performed by x-ray diffraction methods. NMR structures of reduced plastocyanins from a green alga, Scenedesmus obliquus (7, 8), French bean (9), parsley (10), and a blue-green alga Anabaena variabilis (11) have been reported. Poplar plastocyanin is structurally best characterized by x-ray crystallography; structural studies on Hg(II)-substituted plastocyanin (12) and reduced plastocyanin at various pH are reported (13).

At the active site, side chain atoms of the four residues, hystidinyl nitrogens of His37 and His87, a cysteinyl sulfur of Cys84, and a methioninyl sulfur of Met92 coordinate to a copper atom with a distorted tetrahedral geometry. In the oxidized condition, the bond lengths from Cu(II) to these ligands are approximate 1.9 to 2.2 Å except the Cu(II)-Sdelta (Met92) bond of 2.8 to 2.9 Å. Pseudoazurin, one of the blue copper proteins, has a similar coordinational structure to plastocyanin. In pseudoazurin the bond distances from a copper to two histidines and to a cysteine are close to those of plastocyanin and the distance from a copper to a methioninyl sulfur in pseudoazurin is shorter by 0.1 to 0.2 Å than that in plastocyanin.

The strong "blue" absorption band near 600 nm corresponds to the intensed low-energy charge transfer from the Sgamma (cysteine) pi -orbital to the copper dx2-y2 orbital which is the half-occupied redox-active HOMO (14). The copper dx2-y2 orbital lies in the NNS plane formed by the two hystidinyl Ndelta atoms and the cysteinyl Sgamma atom, and the Cu-Sgamma (cysteine) bond bisects the lobes of the orbital (14). Recent studies on the electronic structure of nitrite reductase reported by Solomon and co-workers (15) indicate that the HOMO rotation relative to plastocyanin in the NNS plane decreases the intensity of the blue band. Moreover, the geometry of the copper-thiolate bond will strongly influence the reduction potential and the electron transfer rates through the remote site (14, 15).

The crystal structures of Type 1 blue copper proteins indicate that the oxidized blue copper site is similar to the reduced one. A small change in geometry of copper site gives a small reorganization barrier, which may be preferable to high rate electron transfer (16). The oxidized blue copper site has been considered forced by protein matrix toward the distorted tetrahedral structure which is preferred by Cu(I), because typical Cu(II) inorganic compounds prefer square planar. According to the review described by Williams (17), this idea has led to the theory, the entatic state theory, from the published work by Vallee and Williams (18). They regarded protein as a rigid frame and concluded that Cu(II) ion is enforced into an unusual state by the rigid frame to play a role of electron transfer protein. A similar theory, the induced-rack theory, has been reported by Malmström (19, 20). These theories are based on the idea that the copper-ligand geometry is strained by protein conformation. The difference between the theories is described by Williams (18): the entatic state theory supposes that protein matrix is rigid, while the induced-rack model requires little flexibility of protein structure.

Recently, important studies on the copper-ligand coordination were performed by Ryde et al. (21). They calculated the optimized copper geometry on modeled blue copper proteins and concluded that the results are inconsistent with the entatic state and induced-rack theories. Thus copper-ligand geometry is quite important for functions and spectroscopic properties of blue copper proteins. Here we report a novel insight into the structure of copper site of plastocyanin from the green alga Ulva pertusa.

    EXPERIMENTAL PROCEDURES

U. pertusa Plastocyanin-- The thalli of U. pertusa were harvested at a beach, Futtsu, Chiba, Japan, and plastocyanin was purified by conventional methods (22). The complete amino acid sequence has been determined and will be reported elsewhere.1

Crystallization-- Single crystals of U. pertusa plastocyanin were obtained by the hanging drop vapor diffusion method. The reservoir solution consists of 100 mM glycine buffer (pH 10.0) containing 2.2 M ammonium sulfate. The actual pH of the solution was measured to be 7.6. The protein drop was prepared by mixing 3 µl of protein solution (10 mg/ml) with 3 µl of the reservoir solution. Single cubic crystals appeared within 1 week.

X-ray Analyses and Data Collection-- Preliminary x-ray experiments were performed on a Rigaku R-AXIS IIC imaging plate detector system equipped with a Rigaku RU-300 rotating-anode x-ray generator (fine-focused CuKalpha , operating at 40 kV and 100 mA). Laue symmetry and cell dimensions were determined by the PROCESS program package (23) and systematic absences were confirmed by the pseudo-precession program HKLPLOT in the CCP4 suite (24). The crystal belongs to cubic space group P4132 or P4332 with the unit cell dimension of a = 88.3 Å. Supposing one molecule per an asymmetric unit, a Vm value is calculated to be 2.73 Å3 Da-1 (solvent content 54.9%) (25). Diffraction spots up to 2.0-Å resolution were found on the imaging plates.

Diffraction intensity data were collected by using synchrotron radiation (BL-6B beam line, Photon Factory, KEK, Japan). The Weissenberg camera for macromolecules (26) and large-format imaging plates (40 × 80 cm) (27) were used for the data collection. The crystal diffracts up to 1.6-Å resolution. Fifteen frames with a rotation angle of 2.5° for each frame were stored. Data processing was performed by the program DENZO and SCALEPACK (28). The combined set gave 115,971 reflections to 1.6-Å resolution in total, which were reduced 14,759 unique reflections with an Rmerge of 8.2% and a completeness of 91.5% (70.2% for 1.60 to 1.66 Å). The I/sigma (I) value is 2.3 in the highest resolution shell.

Structural Analysis and Refinement-- Structure analysis was performed by the molecular replacement method. The crystal structure of E. prolifera plastocyanin (5) with an 85% homology to U. pertusa plastocyanin was used as a search model. The amino acid sequence of U. pertusa plastocyanin (Fig. 1) is identical to Ulva arasakii plastocyanin (22) except for residue 12 (correspond to residue 11 of poplar plastocyanin): Ala12 in U. arasakii plastocyanin is substituted to a serine in U. pertusa plastocyanin (Fig. 1).1 Fourteen nonidentical residues were changed to an alanine to improve the signal to noise ratio. Rotation and translation searches were run using the program X-PLOR (29). The diffraction data of 15.0-4.0-Å resolution were used for these calculations. The Patterson correlation refinement (29) indicates that the seventh peak of the rotation function (theta 1 = 313.1°, theta 2 = 46.4°, theta 3 = 239.5°) is the correct solution. The translation searches were performed in the both space groups P4132 and P4332. A significant peak was obtained from the calculation in the space group P4332 (x = 0.759, y = 0.315, z = 0.111). The height of the top peak was T = 0.5731 for P4332 and T = 0.2775 for P4132. An R-factor in P4332 was 0.44 for a 15.0-4.0-Å resolution shell.


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Fig. 1.   Amino acid sequences of plastocyanin from U. pertusa,1 E. prolifera (52), C. reinhardtii (53), S. obliquus (7, 8), Chlorella fusca (54), A. variabilis (55), Synechocystis PCC6803 (56), Silene latifolia (57), Arabidopsis thaliana (58), Spinacia oleracea (59), Populus nigra a (3), Pisum sativum (60), Nicotiana tabacum a (61), Lycopersicon esculentum (62), Phaseolus vulgaris (63), Cucurbita pepo (64), Petroselinum crispum (65), and Prochlorothrix hollandica (66).

Amino acid residues were replaced by using the program TURBO-FRODO (30). A 2Fo - Fc electron density map clearly indicates the structures of the missing side chains. These side chains were fitted to the 2Fo - Fc electron density map. In the first step of structural refinement, rigid-body refinement, positional refinement, and individual temperature factor refinement were applied, and an Rwork decreased from 0.452 to 0.210 for an 8.0-2.3-Å resolution shell. In the third refinement step, 39 water molecules were picked from an Fo - Fc difference Fourier map. The Rwork was decreased to 0.173 in this step. Gradually, the resolution range was expanded and the 10.0-1.6-Å resolution data were included at the 15th step. Three further refinement steps gave the final model. In all refinement steps, force constants for the copper-ligands bonds were set at 50 kcal/mol Å-2 and the equilibrium bond lengths were set as follows: Cu-Ndelta (histidines), 2.10 Å; Cu-Sgamma (cysteine), 2.10 Å; Cu-Sdelta (methionine), 2.80 Å, where the Cu-Sdelta (methionine) distance is an average of plastocyanins and pseudoazurins. The results of the refinement are summarized in Table I.

The structural refinement was also performed using the SHELX program package to judge the accuracy of the final model and to prevent a program-dependent local minimum. The resolution range and the atoms subjected to the refinement are same as X-PLOR. No restraints were applied at the copper site. Three refinement steps including manual adjustment of the model were accomplished. At each step, the coordinate shifts after 10 cycles of a conjugate-gradient least square minimization were within 0.03 Å for the copper atom and its ligands, and changes of each copper-ligand distance were within 0.02 Å. All copper-ligand parameters resulted from the SHELX refinement were close to those from X-PLOR.

    RESULTS AND DISCUSSION

Quality of the Structural Model-- The crystal structure of U. pertusa plastocyanin was determined and refined to a crystallographic R-factor of 0.176 and an Rfree of 0.211 at 1.6-Å resolution (Table I). The final model has good stereochemistry with root mean square (r.m.s.)2 deviations of 0.012 Å from the ideal bond lengths and 5.6° from bond angles (Table I). Except for glycines and prolines, 75 residues are found in the energetically most favored regions and the other 5 residues are found in additional allowed regions (31). All glycines and prolines also have favorable dihedral angles. From the Luzzati plot (32), the upper error limit of the atomic coordinates was estimated to be between 0.20 and 0.25 Å.

                              
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Table I
The refinement statistics

Overall Structure and Comparison with Other Plastocyanins-- U. pertusa plastocyanin has the eight-stranded beta -sandwich structure (5), which is identical to the plastocyanins from poplar, C. reinhardtii, and E. prolifera. The beta -sandwich can be divided into two beta -sheets, beta -sheet I and beta -sheet II, as described by Freeman and co-workers (5). The beta -sheet I consists of four beta -strands: S1 (residues 1 to 5), S2A (14 to 16), S3 (26 to 31), and S6 (69 to 73) (Fig. 2). The beta -sheet II also contains four beta -strands; S2B (residues 18 to 22), S4 (39 to 43), S7 (78 to 83), S8 (93 to 99). The copper site is located at a northern end of the molecule (Fig. 2). Seven turns, turns 1-7, were found. Turn 2 is a type IVb turn (33) with no hydrogen bond and is bent at cis-Pro16. Two turns, turn 3 and turn 5, are type II turns and the others are type I turns. Turns 6 and 7 are located around the copper site. Hydrogen bonds in the backbones of turns 6 and 7 are essential for maintaining the structure of the copper site.


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Fig. 2.   Schematic drawing of U. pertusa plastocyanin. The copper atom is shown in cyan at the top of the model. The four ligands, His37, Cys-, His87, and Met92, are represented by ball-and-stick models. Color configuration of the ligands are similar to that of Fig. 4. The names of the eight beta -strands are shown in the figure. This figure was drawn by the program PROTEUS (System Co., Ltd., Japan).

r.m.s. deviations of the structures between the main chain atoms of U. pertusa plastocyanin and those of the others are as follows: poplar, 0.74 Å; C. reinhardtii, 0.62 Å; E. prolifera, 0.43 Å. Large deviations were found at four regions, around residues 26, 55, 70, and 87 (Fig. 3). The large deviations at the second region are responsible for the two inserted residues, 58 and 60, in poplar plastocyanin. The fourth region contains turns 6 and 7, in which Cys84, His87, and Met92 construct a part of the copper site (Fig. 4). The structural difference of this site influences the structure of the copper site and we will discuss it in the next section.


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Fig. 3.   r.m.s. differences along the main chain atoms (N, Calpha , C, and O) between U. pertusa plastocyanin and the others, poplar (thin solid line), C. reinhardtii (thin broken line), and E. prolifera (thick solid line).


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Fig. 4.   Ball-and-stick models at the copper site with a 2Fo - Fc electron density map. The map was contoured at a level of 1.5 sigma .

Comparison of the Copper Site with Other Plastocyanins and Blue Copper Proteins-- In plastocyanins, Cu(II) is coordinated by four atoms, Ndelta atoms of His37 and His87, the Sgamma atom of Cys84, and the Sdelta atom of Met92, in a distorted tetrahedral geometry (Fig. 4). The bond distances and the bond angles of the copper site are summarized in Table II. In U. pertusa plastocyanin, the bond distance between Cu(II) and Sdelta (Met92) is shorter by 0.13 to 0.23 Å than the other plastocyanins and is rather close to that of pseudoazurin and nitrite reductase (Table II). This short Cu(II)-Sdelta (Met92) distance can be explained by the structural differences at turn 7 (residues 88 to 91). The large deviation is found at turn 7 located near the copper site (Figs. 3 and 5). At turn 7, the amino acid difference is found at only residue 88 (Fig. 1): U. pertusa has an alanine; E. prolifera has a serine; poplar and C. reinhardtii have a glutamine. E. prolifera plastocyanin has a hydrogen bond between O(Asp85) and Ogamma (Ser88) (Fig. 5A), which withdraws turn 7 toward Asp85. The Gln88 residue of poplar and C. reinhardtii plastocyanins do not have the hydrogen bond but a cation-pi interaction (34, 35) with Tyr83 by stacking on its an aromatic ring (Fig. 5B), which is the "remote site" residue. The O(Asp85)-Ogamma (Ser88) hydrogen bond found in E. prolifera plastocyanin pulls the Calpha (Met92) atom toward Asp85 through the backbone of the turn 7 (Fig. 5A). Compared with U. pertusa plastocyanin, Met92 of E. prolifera plastocyanin rotates 12° around its Calpha atom, and Calpha (Met92) atom translates by 0.4 Å. This effect lengthens the Cu(II)-Sdelta (Met92) distance in E. prolifera plastocyanin. Corresponding regions of poplar and C. reinhardtii plastocyanins, with the cation-pi interaction between Tyr83 and Gln88, have an intermediate structure between U. pertusa and E. prolifera. In conclusion, the Cu(II)-Sdelta (Met92) distance is strongly influenced by the structure of turn 7: U. pertusa plastocyanin with the little restraint has the shortest distance of 2.69 Å, E. prolifera plastocyanin with the strong restraint has the longest distance of 2.92 Å, and poplar and C. reinhardtii plastocyanins with the weak restraint have intermediate distances of 2.82 and 2.89 Å, respectively.

                              
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Table II
Bond lengths and bond angles of copper site
The resolution limit and R-factor of each protein are as follows: poplar, 1.33 Å, R = 0.150; C. reinhardtii, 1.5 Å, R = 0.168; E. prolifera, 1.85 Å, R = 0.117; Pseudoazurin, 1.5 Å, R = 0.199; NiR, 1.9 Å, R = 0.168


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Fig. 5.   Comparison of the structures of plastocyanins around the turn 7 and the copper site. U. pertusa plastocyanin (yellow) is superimposed against E. prolifera (red) in A and poplar plastocyanin (orange) in B. Green dots mean the Ogamma (Ser88)-O(Asp85) hydrogen bond in E. prolifera plastocyanin. The models are translated after the r.m.s. fitting so that the copper atoms of all models occupy the same position. To clarify the view, the following atoms are omitted: side chain atoms of residues 85, 86, 89, and 90; carbonyl oxygen atoms of residues 83, 84, and 86-92. This drawing was prepared by the program MIDASPLUS (67, 68).

Ryde et al. (21) calculated the energy of the copper site as a function of Cu(II)-Sdelta (methionine) distance (21). According to their results, energies of the copper sites of U. pertusa and E. prolifera plastocyanins are higher by approximately 1 and 5 kJ/mol, respectively, than the minimum energy point. An energy of the copper site of E. prolifera plastocyanin is then higher by approximately 4 kJ/mol than that of U. pertusa plastocyanin, taking account of only the Cu(II)-Sdelta (Met92) distance. This energy difference fits surprisingly well to the binding energy of the O(Asp85)-Ogamma (Ser88) hydrogen bond in E. prolifera plastocyanin if we suppose that the effect of the hydrogen bond is 0.5 to 1.5 kcal/mol (correspond to 2.2 to 6.5 kJ/mol) as reported by Fersht et al. (36). In the case of poplar and C. reinhardtii plastocyanins, cation-pi interaction between Tyr83 and Gln88 are estimated at about 2 and 4 kJ/mol, respectively. These agree with the binding energy (~10 kJ/mol) of the benzene-ammonia system (37) which is a model of cation-pi interaction with a neutral nitrogen. Thus, the shorter Cu(II)-Sdelta (Met92) distance of U. pertusa plastocyanin due to the absence of the O(Asp85)-Ogamma (Ser88) hydrogen bond is consistent with the results of the energy analysis on the Cu(II)-Sdelta (methionine) coordination. In conclusion, U. pertusa plastocyanin, free from restraint by the hydrogen bond between residues 85 and 88, is close to the minimum energy state, whereas E. prolifera plastocyanin, with the elongated Cu(II)-Sdelta (Met92) bond by the O(Asp85)-Ogamma (Ser88) hydrogen bond, has the higher energy state. Consequently, our crystal structure suggests that the Cu(II)-Sdelta (Met92) bond of E. prolifera plastocyanin is slightly "racked" by the O(Asp85)-Ogamma (Ser88) hydrogen bond.

U. pertusa plastocyanin has the longest Cu(II)-Sgamma (Cys84) distance in all reported crystal structures of plastocyanins (Table II). This value is rather close to that of pseudoazurin and nitrite reductase compared with those of plastocyanins. The optimized Cu(II)-Sgamma (cysteine) distance (Table II) calculated by the quantum chemical theories is in good agreement with U. pertusa plastocyanin, pseudoazurin, and nitrite reductase, whereas the other plastocyanins deviate significantly from the optimized distance as follows: poplar, shorter in 0.11 Å; E. prolifera, shorter in 0.06 Å; C. reinhardtii, shorter in 0.07 Å (Table II). The shorter Cu(II)-Sgamma (cysteine) distance is compensated by the longer Cu(II)-Sdelta (methionine) distance (15). Therefore, the longer Cu(II)-Sgamma (Cys84) distance of U. pertusa plastocyanin may be responsible for the shorter Cu(II)-Sdelta (Met92) distance.

In addition, evidence for the longer Cu(II)-Sgamma (Cys84) distance of U. pertusa plastocyanin was found by the resonance Raman spectrum (38). The Raman band corresponding to Cu(II)-Sgamma (Cys84) bond of U. pertusa plastocyanin is observed at lower frequency than the others. This result strongly supports the present results.

Structural Basis for Regulation of the Blue Copper Site by Residue 88-- The present work first provides experimental proof that the copper geometry is sensitive to the structure of the loop between His87 and Met92 (His-Met loop) (corresponding to turn 7 in U. pertusa plastocyanin). This means that the copper site of the blue copper proteins is more flexible than reported formerly (17-20). However, it should be noted that the copper site is strained even if its effect is weak. It is impossible to control the copper geometry by the His-Met loop, if it were rigid. Flexibility of the copper site, especially that of Cu-Sdelta (methionine) bond, should be required for the structure of plastocyanin. Consequently, the present results indicate that the rack model (19, 20), which agrees with the softness of the blue copper site, is more plausible for the blue copper proteins rather than the entatic theory (17, 18).

In the case of azurin, the fifth ligand of a carbonyl oxygen coordinates to the copper atom with a distorted trigonal bipyramidal shape (39-43). Compared with the blue copper proteins with distorted tetrahedral geometry, the Cu-Sdelta (methionine) distance of azurin is long by approximately 0.3 to 0.5 Å. In azurin, the weak Cu-O bond attracts the copper ion to the ligated oxygen and causes the lengthening of Cu-Sdelta (methionine) bond. This is consistent with the soft interaction of the methioninyl sulfur with the copper ion and suggests that the Cu-O bond is well balanced against the soft Cu-Sdelta (methionine) bond in azurin.

Strength of the interaction between residues 85 and 88 is quite important to control the Cu-Sdelta (methionine) geometry, because the copper geometry is crucial for the electron transfer ability. In the His-Met loop, the discrepancy of primary structures is only found at residue 88 (Fig. 1) in various sources. This implies that the conserved residues in the loop are essential to maintain its structure, whereas residue 88 probably characterizes the properties of plastocyanin. The structural diverseness of the His-Met loop caused by the difference of residue 88 may vary the electron transfer rate in plastocyanin and other blue copper proteins.

Residue 88 which controls the structure of the His-Met loop is close to Tyr83 which is in the negative or acidic patch and is one of the electron transfer paths. The electron transfer reaction of plastocyanin with cytochrome f (44) or P700 (45) should occur when plastocyanin forms the complex with the negative patch. Residue 88 in the His-Met loop will then change its conformation by interacting with the pair, which will modify the geometry of the copper site through the loop. The complex should form the most appropriate conformation to reach the most efficient electron transfer reaction. Consequently, the geometry of the blue copper center may transform into the most suitable form by the conformational change of the loop.

In the Marcus theory (16), an electron transfer rate reaches its maximum value when the nuclear factor is optimized (Delta G° lambda , where Delta G° and lambda  are the driving force and the reorganization energy, respectively). Moreover, the conformational change is an important factor for the reorganization energy in a protein (46). Within the complexes between plastocyanin and its pair, the most important conformational change may occur probably at the His-Met loop and the copper site. That is to say, to reach the optimized condition of the nuclear factor, the conformational change of the His-Met loop occurs when the complex is formed, and then it may optimize the structure of the copper site.

    ACKNOWLEDGEMENTS

We thank Professor N. Sakabe, Dr. N. Watanabe, and Dr. M. Suzuki for support in data collection at KEK, Japan.

    FOOTNOTES

* This work was supported in part by 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 coordinates and the structure factors (code 1IUZ) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Dagger Present address: Dept. of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigori, Ako-gun, Hyogo 678-1297, Japan.

parallel 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.

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

1 T. Fukazawa, unpublished results.

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Abstract
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