Crystal Structure Determinations of Oxidized and ReducedPseudoazurins from Achromobacter cycloclastesCONCERTED MOVEMENT OF COPPER SITE IN REDOX FORMS WITH THE REARRANGEMENT OFHYDROGEN BOND AT A REMOTE HISTIDINE*

(Received for publication, February 3, 1999, and in revised form, April 8, 1999)

Tsuyoshi Inoue, Nobuya Nishio, Shinnichiro Suzuki‡, Kunishige Kataoka‡,Takamitsu Kohzuma§, and Yasushi Kai¶

From the Department of Materials Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871,Japan, the ‡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

The crystal structures of oxidized and reducedpseudoazurins from a denitrifying bacterium, Achro-mobacter cycloclastes IAM1013, have been determined at1.35- and 1.6-Å resolutions, respectively. The copper sitein the oxidized state exhibits a distorted tetrahedralstructure like those of other pseudoazurins. However,not only a small change of the copper geometry, butconcerted peptide bond flips are identified. The imidaz-ole ring of remote His6 has a hydrogen bonding distanceof 2.73 Å between N-d1(His6) and O-g1(Thr36) in the oxi-dized protein. When the protein is reduced at pH 6.0, theimidazole ring rotates by 30.3° and moves 1.00 Å awayfrom the position of the oxidized state. A new hydrogenbond between N-e2(His6) and O-e1(Glu4) is formed with adistance of 3.03 Å, while the hydrogen bond betweenN-d1(His6)-O-g1(Thr36) is maintained with an inter-atomic distance of 2.81 Å. A concomitant peptide bondflip of main chain between Ile34 and Thr36 occurs.

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 elec-tron transfer substituted to another blue copper protein namedamicyanin at high copper concentration. The ternary complexamong methylamine dehydrogenase, amicyanin, and cyto-chrome c551 shows that amicyanin accepts an electron frommethylamine dehydrogenase (1). On the other hand, in deni-trifying bacteria pseudoazurins donate an electron for the re-duction of NO2

2 to NO by nitrite reductase under anaerobicconditions (2–5). Pseudoazurins from four sources, Achro-mobacter cycloclastes IAM1013 (2, 6, 7), Methylobacterium ex-torquens AM1 (8), Alcaligenes faecalis S-6 (3, 9), and Thiospha-era pantotropha (10) have been characterized to date. Fromtheir amino acid sequences, it is known that A. cycloclastespseudoazurin has an extra C-terminal residue giving 124

amino acids in total (11). The greatest degree of similarityamong pseudoazurins is 65% conservation of amino acid resi-dues between A. cycloclastes and A. faecalis.

The crystal structure analyses of pseudoazurins from A. fae-calis (11–14), T. pantotrophs (15), and M. extorquens (16) re-vealed that the overall topology of pseudoazurin consists of aneight-stranded b-barrel which resembles the structures ofother cupredoxins like plastocyanin and azurin. Differencesoccur between these proteins regarding the amount and loca-tion of helical structure. Pseudoazurin possess two extra a-hel-ices at the C terminus, whereas azurin has an a-helical flap inthe middle of the sequence and plastocyanin generally has asmall (1 turn) helix (17). The copper atom is located below theprotein surface at a depth of 5–10 Å, with two histidines (im-idazole N), a cysteine (thiolate RS-), and methionine (thioetherS) as ligands (18). Nine of 13 lysine residues surrounded thehydrophobic face of the molecule through which the histidineligand protrudes slightly. The effect of lysine residues for theelectron transportation from pseudoazurin to nitrite reductasewas well investigated by using mutant pseudoazurin from A.faecalis S-6 (19). The substitution of these basic lysine residuesby amino acids with neutral/acidic side chains decreases theaffinity of pseudoazurin for nitrite reductase, which suggeststhat pseudoazurin may interact with nitrite reductase throughits hydrophobic patch. Moreover, the structure of pseudoazurinfrom T. pantotrophs revealed that the proposed docking motifsbased on the positive hydrophobic surface patch for cytochromecd1 nitrite reductase (15).

Small inorganic complexes such as [Fe(CN)6]32 and [Co-(phen)3]31 (phen 5 1,10-phenanthroline) have been used asredox probes to identify potential binding sites on the surface ofcupredoxins for electron-transfer partners (20). Recent kineticand theoretical studies on the blue copper protein plastocyaninhave indicated the presence of two distinct electron-transfersites: (i) the adjacent hydrophobic patch ;6 Å from the copperthrough which one of the histidine ligands protrudes, and (ii)the remote site involving and acidic patch region ;15 Å fromthe copper, with acidic residues on either side of the exposedTyr83 (Tyr83 is next to the copper ligand Cys84). Kinetics stud-ies with small inorganic complexes have been carried out on A.cycloclastes pseudoazurin. The rate constant for the oxidationof the reduced molecule with [Fe(CN)6]32 is 103-fold biggerthan that for the [Co(phen)3]31 oxidation (21), indicating aclear preference of the protein for anionic species. Moreover,the self-exchange rate constant for reduced molecule (about 3 3103 M21 s21) is much smaller than those of most other cupre-doxins and is quite similar to that found in higher plant plas-

* This work was supported in part by Grant-in-Aids 09261223,03241215, 04225216, 05209216, and 08249221 for Scientific Researchon 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 AdvancedResearch Alliance (TARA) Sakabe project. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 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 NationalLaboratory, Upton, NY.

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

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 25, Issue of June 18, pp. 17845–17852, 1999© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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tocyanins (22). A. cycloclastes nitrite reductase, which pos-sesses many acidic amino acid residues, accepts an electronfrom a reduced pseudoazurin with a second-order rate constantof 7–9 3 105 M21 s21 (5, 23). These findings are all probably due

to the effect of a number of conserved lysine residues surroundthe hydrophobic patch of pseudoazurin.

The effect of pH on the redox reactivity of A. cycloclastespseudoazurin is well documented (21, 22, 45). The reduced

FIG. 1. Ribbon presentation of the oxidized pseudoazurin from A. cycloclastes viewed from the side of the molecule (a) and fromabove 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, arerepresented by ball-and-stick model. The uncoordinated His6 and basic residues (Arg and nine Lys) surrounding the hydrophobic surface are alsoshown with ball-and-stick. The figure is drawn by program MOLSCRIPT (43) and RASTER3D (44).

TABLE IData collection and final refinement statistics for the oxidized and reduced pseudoazurins from Ach. cycloclastes

Oxidized form Reduced form

Data collectionSource Synchrotron CuKaCamera Weissenberg Camera (BL-6A) RAXIS-IIcCrystal to detector distance 286.5 65.0No. of crystal 2 1Resolution range (A) 30.0–1.35 30.0–1.6Reflections measured/unique 22,2685/21,303 58,870/13,287Completeness (%) overall/outer shell 88.8/56.0 90.4/70.8

(1.40–1.35A) (1.66–1.60A)Rmerge overall/outer shell 8.5/18.2 4.0/14.1

(1.40–1.35A) (1.66–1.60A)Final refinement statistics

Resolution (Å) 8.0–1.35 8.0–1.6No. of atoms 913 913No. of copper ion 1 1No. of water molecules 121 .96No. of reflections

Working set 20,012 12,449Test set for Rfree 1,084 680

R (%) 17.6 17.3Rfree (%) 19.0 21.1Average temperature factors (A2)

All atoms 11.3 18.2Main chain 8.2 15.3Side chain 11.5 19.0Metal ion 5.4 15.9Solvent atoms 23.5 29.9

r.m.s. deviations from standardgeometries

In bond length (A) 0.014 0.016In bond angles (°) 2.38 2.5

Ramachandran plotMost favored (%) 92 92Allowed 8 8

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protein has been shown to participate in an active site proto-nation/deprotonation equilibrium at His81, which gives an aciddissociation pKa of 5.2 from an NMR titration (21) and 4.6 fromkinetic studies with inorganic complexes as oxidants (45). Theeffect of protonation/deprotonation of uncoordinated His6 resi-due located near to the active site shows a pKa values of 7.2(reduced form) and 6.5 (oxidized form) from NMR titration and7.3 from kinetic studies of the oxidation of the reducedpseudoazurin with small complexes (21).

The structure of reduced pseudoazurin from A. faecalis at pH4.4 has been solved and demonstrates that the ligand His81

rotates away from the metal due to protonation of the N-d1atom (14). Small changes were observed in the copper vicinityand on the protein surface at pH 7.8 upon reduction (14), whiledistinct conformational changes occurred in response to reduc-tion 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 pseudoa-zurin were reported by Turley et al. (25), but the crystalsobtained were too thin for x-ray diffraction data to be obtained.We have previously reported the crystallization and prelimi-nary x-ray studies on oxidized pseudoazurin from A. cyclo-clastes at pH 6.0 (26). In this work single crystals of oxidizedand reduced pseudoazurin with high resolutional diffractionspots were obtained at pH 6.0. During the last stage of struc-ture refinement, the amino acid sequence was partially cor-rected (27).1 We describe here the redox-induced conforma-tional changes at the copper site, and the rearrangement of thehydrogen bonding pattern of the uncoordinated His6, which arequite similar, but not identical, to the pattern of changes foundin A. faecalis pseudoazurin reduced at pH 7.0 (24). The peptideflip induced by protonation at His6 is pH-induced conforma-tional transition of His35 in azurin from Pseudomonasaeruginosa (28).

EXPERIMENTAL PROCEDURES

Data Collection and Processing—The crystallization (0.1 M potas-sium phosphate buffer (pH 6.0)) and structural analysis of oxidizedpseudoazurin from A. cycloclastes were performed as described previ-ously (26). The data with high resolution was collected with synchrotronradiation at the BL6A2 station of 2.5 GeV energy produced by thestorage ring in the Photon Factory, the National Laboratory for HighEnergy Physics (KEK), Tsukuba, Japan. Diffraction patterns were re-corded on a Fuji Imaging Plate (200 3 400 mm, Fuji Photo Film) (29)using Sakabe’s Weissenberg camera for macromolecules (30) with anaperture collimator of 0.1-mm diameter and a cylindrical cassette of286.5-mm radius filled with helium gas. The intensity data were pro-cessed using DENZO and scaled with the program SCALEPACK (31).Among 222,685 accepted observations up to 1.35-Å resolution, 21,303independent reflections were obtained, the completeness of which was88.8% with an Rmerge value of 8.5%. The reduced crystals of pseudoa-zurin were obtained by soaking the oxidized crystals with the crystal-lization solution (0.1 M potassium phosphate, pH 6.0) containing 10 mM

sodium L-(1)ascorbate. After 10 min the blue crystals became colorless.For more than 1 month the crystals remained colorless in the glasscapillary with the mother liquor containing 10 mM sodium L-(1)ascor-bate. The x-ray diffraction data up to 1.6-Å resolution were obtainedusing the imaging plate detector operated in the Rigaku RAXIS-IIcsystem. Among 58,870 accepted observations up to 1.60-Å resolution,13,287 independent reflections were obtained, the completeness ofwhich was 90.4% with a Rmerge values of 4.0%.

Structure Analysis and Refinement—Structure analysis has beencarried out by the molecular replacement method using the MERLOTprogram package (32). Calculations using the molecular structure ofpseudoazurin from A. faecalis as a starting model were performed. The

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

FIG. 2. Superimposition of the oxidized (thin lines) and re-duced (thick lines) pseudoazurins from A. cycloclastes for wholestructures (a) and the copper sites (b). A quite small r.m.s. devia-tion of 0.14 Å is calculated for whole structures. However, the coppersite and the uncoordinated His6 are significantly moved on reduction atpH 6.0.

TABLE IIMetal coordinations of pseudoazurins

Distance parameters (Å)

N-d of His(1)

S-g of Cys(2)

N-d of His(3)

S-d of Met(4) Oxygen atom

A. cycloclastes (oxidized, pH 6.0) 1.95 (H40) 2.13 (C78) 1.92 (H81) 2.71 (M86) 3.94 (G39)A. cycloclastes (reduced, pH 6.0) 2.04 (H40) 2.19 (C78) 2.11 (H81) 2.85 (M86) 4.04 (G39)Al. faecalis S-6 (oxidized, pH 6.8) 2.20 (H40) 2.14 (C78) 2.27 (H81) 2.67 (M86) 3.99 (G39)Al. faecalis S-6 (reduced, pH 7.8) 2.16 (H40) 2.17 (C78) 2.29 (H81) 2.91 (M86) 3.83 (G39)Al. faecalis S-6 (reduced, pH 4.4) 2.19 (H400 2.16 (C78) 3.09 (H81) 2.42 (M86) 4.01 (G39)M. extorquens AM1 (oxidized, pH 8.0) 2.07 (H40) 2.15 (C78) 1.97 (H81) 2.66 (M86) 4.03 (G39)

Angle parameters (Å)

(1)-Cu-(2) (1)-Cu-(3) (1)-Cu-(4) (2)-Cu-(3) (2)-Cu-(4) (3)-Cu-(4)

A. cycloclastes (oxidized, pH6.0)

107 135 100 87 114 107

A. cycloclastes (reduced, pH 6.0) 132 102 90 116 107 104Al. faecalis (oxidized, pH 6.8) 138 100 86 111 107 109Al. faecalis (reduced, pH 7.8) 140 102 85 108 107 110Al. faecalis (reduced, pH 4.4) 138 94 97 83 122 116M. extorquens (oxidized, pH 8.0) 144 94 86 110 109 112

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highest rotation peak was found at Eulerian angles of a 5 115.0°, b 560.0°, and g 5 305.0° for 13–3.2-Å resolution data. The translationparameters were calculated in the RVAMAP function with MERLOT,and the highest peak (x 5 0.45, y 5 0.10, and z 5 0.15 in fraction of unitcell) gave the minimum R-factor of 64.9% for 15–3.88-Å resolution data.After several cycles of the RMINIM function, the rotation parametersconverged to a 5 115.40°, b 5 62.23°, and g 5 303.99°, and the trans-lation parameters to 3 5 0.4537, y 5 0.1330, and z 5 0.1387. TheR-factor calculated for the model structure was 47.5% for 15.0–3.88-Ådata. Refinements for both oxidized and reduced structures have beencarried out by using PROLSQ: (33), X-PLOR version 3.0 (34), andTURBO-FRODO (35). The first step of the refinement for both formswas performed using X-PLOR. A simulated annealing calculation at3000 K, further several steps of positional refinement were performedby X-PLOR using the stepwise increased data. Because of the ambiguityfor the energy constraint parameters of metal atom used in the molec-ular dynamics refinement, somewhat unreliable copper geometrieswere obtained. In order to obtain exact copper geometry no restraintwas imposed on the copper coordination through the final stage ofrefinement by REFMAC. Every stage includes model improvementfollowed by several cycles of refinement. Model improvements werecarried out based on the Fourier maps calculated with the coefficients of(2Fo 2 Fc)exp(2piacalc) and (Fo 2 Fc)exp(2piacalc). The final structuremodel, further rebuilding, and cycles of refinement for the water struc-ture of oxidized molecule finally decreased the R-factor to 17.6% for thedata between 8.0- and 1.35-Å resolution with quite reasonable stereo-chemistry. 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 forthe reduced structure by using the data between 8.0- and 1.6-Å resolu-tion were 17.3 and 21.1%. The results of data collection and refinementare summarized in Table I.

RESULTS

Quality of the Final Model—The final model of oxidizedpseudoazurin from A. cycloclastes is made up of one monomerin the asymmetric unit with 124 amino acids, 121 water mol-ecules, and single copper ion. The model remained close tostandard geometry throughout refinement. The final model ofreduced structure includes 96 water molecules. The mean po-sitional errors of the atoms estimated by Luzzati plots are0.137 Å for the oxidized protein and 0.153 Å for reduced form,respectively (38). For well defined parts of the structure, espe-cially the b-strands, the internal side chains and the regionaround the metal site, the errors are likely to be lower. Thequality of the final model is summarized in Table I. The pro-gram PROCHECK (39) was used to analyze conformationalvariations from defined norms. A Ramachandran plot (40)shows that all non-glycine residues have dihedral angles fallingin (or near to) energetically preferred regions.

Overall Structure—A ribbon drawing of A. cycloclastespseudoazurin is presented in Fig. 1. The approximately spher-

FIG. 3. The oxidized (a) and reduced (b) structures of pseudoazurin from A. cycloclastes with omit maps. Structural comparisonaround copper site and His6 between the oxidized (line in blue) and reduced (line in magenta) forms (c). The copper coordination structure ischanged from blue line to yellow line on reduction. Concerted movements around His6 at the distance of 12 Å from the copper ion are shown inball-and-stick models colored in blue (oxidized form) and red (reduced form). The dihedral angle between the hydrogen bond of N-d1(His6)-O-g1(Thr36) and the imidazole ring was changed from 0.24° to 230.1° upon reduction. Namely, the imidazole ring moved through a distance of 1.00Å and rotated by 30.3° against the relative position of O-g1(Thr36). On the contrary, the distance of O-e2(Glu4)-N-e2(His6) is changed from 3.47 Åto 3.07 Å and a new hydrogen bonding is formed. C-a(Pro35) and C-g(Pro35) moved 0.93 Å and 2.16 Å, respectively, and the dihedral angle of Ca-Cb

and Cg-Cd in Pro35 was changed by 30.9°. The water molecule (green color), which locates in the oxidized state with a temperature factor of 13.9Å2 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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ical pseudoazurin molecule has overall dimensions of 38 3 38 327 Å. The molecule possesses eight b-strands, forming twob-sheets, and two C-terminal a-helices. b-Sheet I consists offour b-strands: S1, residues 2–8; S2a, 17–19; S3, 30–34; S6,

64–67, and b-sheet II contains five b-strands: S2b, residues22–25; S4, 42–44; S5, 56–58; S7, 72–77; S8, 87–92. Thesestructural features are very similar to those of the otherpseudoazurins (from A. faecalis, M. extorquens, and T. panto-

FIG. 3—continued

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tropha) (11–16). The number of identical residues are 81/124(65%) between pseudoazurins from A. cycloclastes and A. fae-calis, 64/123 (52%) between pseudoazurins from A. cycloclastesand M. extorquens, and 54/123 (44%) between pseudoazurinsfrom M. extorquens and A. faecalis. The three structures are soanalogous, except at the C-terminal a-helices, that averagedr.m.s.2 deviations of the backbone structures among threepseudoazurins are less than 0.7 Å (0.66 Å, 0.55 Å, and 0.69 Å,respectively).

Conformational Changes in the Copper Vicinity—A dramaticchange at the copper center, from tetrahedral to trigonal, wasobserved 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 pseudoazur-ins from A. faecalis were observed at pH 7.8 (14), while shiftsaround the copper ion were observed upon reduction at pH 7.0(24). For A. cycloclastes pseudoazurin, the r.m.s. deviation ofbackbone structures between the two oxidation states of theprotein is 0.14 Å (Fig. 2a). The conformational differences of thecopper geometries in the oxidized and reduced forms are shownin Fig. 2b. The copper-His81 distance is significantly length-ened by the reduction of the copper center (0.19 Å) in contrastto the small lengthening of other bonds (copper-His40 (0.09 Å),copper-Cys78 (0.06 Å), and copper-Met86 (0.14 Å)). The cuprouscenter also has a distorted tetrahedral geometry. The bondlengths and angles at the copper centers are summarized inTable II.

Other Redox Linked Conformational Changes—Fig. 3, a andb, show that both models are well fitted to the electron densitymaps. The structure comparison between the oxidized form andthe reduced form is performed by least square method with allbackbone atoms. Despite the quite small r.m.s. deviation of0.14 Å for the backbone, some significant differences are found.The striking finding is the rearrangement of hydrogen bondinginteractions of the non-coordinated His6 and the peptide bondflip around Pro35 as shown in Fig. 3c. The N-d1(His6) forms ahydrogen bond to O-g1(Thr36) with a distance of 2.73 Å in theoxidized pseudoazurin. Both the O-g1(Thr36) and theN-d1(His6) atoms possess relatively low temperature factors of14.4 and 16.1 Å2, respectively. However, the imidazole ring ofHis6 moves through a distance of 1.00 Å and the dihedral anglebetween the hydrogen bond and the imidazole ring changes by30.3° on reduction at pH 6.0. Accompanying this change theN-e2 of His6 to O-e2 of Glu4 distance decreases from 3.47 Å to

3.07 Å forming a new hydrogen bond (Fig. 3c). Apparently thehydrogen bonding between His6 and Thr36 is weakened. Therearrangement of hydrogen bond of His6 leads to a concomitantIle34-Thr36 main chain peptide bond flip. The main chain con-formational angles are also changed dramatically, particularlyaround Pro35 (Table III). The Ca and Cg atoms of Pro35 havemoved by 0.93 and 2.16 Å, respectively.

Loss of Water Molecules Adjacent to Gly39—In azurin thebackbone carbonyl oxygen of Gly45 provides a second weaklyinteracting axial ligand at a distance of 3.1 Å from Cu21,resulting in a distorted trigonal bypyramidal coordination ge-ometry. In plastocyanins and pseudoazurins the correspondingdistance is longer than that in azurin, and the active sitegeometry is distorted tetrahedral. The O(Gly39) of A. cyclo-clastes pseudoazurin, which corresponds to O(Gly45) in azurins,locates at a distance of 3.94 Å from the copper ion in theoxidized protein, and a water molecule with a temperaturefactor of 13.9 Å2 is found at distances of 2.92, 2.83, and 3.04 Åfrom N(His40), O(Asp37), and O(Asn61), respectively (Fig. 3c).Because the O(Gly39) is adjacent atom of N(His40), the watermolecule forms a long range hydrogen-bonding network rang-ing from O(Gly39) to O(Asn61). However, upon reduction thedistance between O(Gly39) and the copper ion is lengthened to4.04 Å, which shows the extension of ionic radius of the coppercenter. Moreover, the water molecule is lost providing space forthe peptide bond flip of Pro35. The conformations of the mainchain around Gly39 is changed as shown in Table III. Theextended ionic radius of copper and the reduction of the coppercharge may influence the water molecule, which may facilitatethe peptide bond flip and the rearrangement of the hydrogenbonding of His6.

Structural Comparison between Pseudoazurins from A. cyclo-clastes and A. faecalis—The oxidized and reduced pseudoazur-ins from A. cycloclastes are superimposed on those from A.faecalis, respectively, with r.m.s. deviations for backbone at-oms of 0.64 and 0.62 Å. The superimposition reveals the re-markable 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 morethan 70°. The imidazole ring rotates to the perpendicular po-sition relative to that in A. faecalis, which enables it to form thestrong hydrogen bond to Thr36 in the oxidized pseudoazurinfrom A. cycloclastes. The residue Glu4 is conserved in bothpseudoazurin but the 36th amino acid residue is Val instead ofThr in A. faecalis pseudoazurin. Only one hydrogen bond istherefore possible between His6 and Glu4. The N-e2(His6)-O-2 The abbreviation used is: r.m.s., root mean square.

TABLE IIIChange of main chain conformational angles

Amino acidf-Angle (degree) c-Angle (degree)

Oxidized Reduced Difference Oxidized Reduced Difference

Phe3 2129 2125 14 160 163 13Glu4 2127 2123 14 137 139 2Val5 2124 2127 23 128 132 14His6 2100 2106 26 138 145 17Met7 2101 2107 26 113 119 16Leu8 2126 2128 22 148 150 12Asn9 270 269 11 230 232 22Thr30 2115 2116 21 145 143 22Val31 2127 2124 13 126 132 16Thr32 2102 2105 23 126 123 23Phe33 293 293 0 114 123 19Ile34 2106 2122 216 128 116 212Pro35 279 274 15 70 96 126Thr36 262 267 25 242 237 15Asp37 2107 2103 14 150 153 13Lys38 279 275 14 157 152 25Gly39 97 103 16 13 4 29His40 2122 2113 19 161 162 11

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e1(Glu4) distances are 2.76 Å (oxidized form) and 2.96 Å (re-duced form) at pH 7.8 (14) in A. faecalis pseudoazurin, whilethe distances are 2.81 Å (oxidized form) and 2.71 Å (reducedform) at pH 7.0 (24). The rearrangement of hydrogen bond ofHis6 was not observed in pseudoazurin from A. faecalis S-6. Onthe other hand, Thr36 fixes the position of His6 in the directionof O-g1(Thr36) atom and the N-e2(His6) is directed toward theside chain of Glu4 in the oxidized form of pseudoazurin from A.cycloclastes. Thus, whether threonine or valine at residue 36 isthe clue to the appearance of redox-induced rearrangement ofhydrogen bonding around His6.

DISCUSSION

According to the detailed structural comparison between theoxidized and reduced pseudoazurin from A. cycloclastes, thedifference in copper-His81 distance is bigger between two formsthan other copper-ligand distances. The expansion of the ionicradius of the copper, 0.96 Å for Cu11 and 0.69 Å for Cu21, andthe reduction of copper charge upon reduction results in theloss of a water molecule situated close to the active site. Forthis reason the peptide bond flip at Pro35 may occur moreeasily. The loss of a similar water molecule was found in A.faecalis pseudoazurin at both pH 7.8 and 7.0, while the peptidebond flip was found at only pH 7.0. Because the pKa values ofthe uncoordinated His6 residue are 7.2 (reduce form) and 6.5(oxidized form) (21), the protonation at His6 may be the causeof the peptide bond flip in Al. faecalis pseudoazurin. A similarpeptide bond flip was observed in oxidized azurin from P.aeruginosa upon changing the pH from 9.0 to 5.5 (28). In thiscase the rearrangement of the hydrogen bonding pattern re-sulted from the protonation/deprotonation of His35 and inducedthe peptide bond flip. The vicinity of the copper site was notsignificantly affected by this conformational change. However,in this study all processes have been performed at pH 6.0, bothforms have the protonated His6. The protonation may not bethe direct trigger of the peptide bond flip in this study. Actu-ally, the strong hydrogen bond between His6 and Thr36 isweakened, and a new hydrogen bond between His6 and Glu4 isformed on reduction. The N-e2 atom of His6 should carry aproton on reduction because it can only be a proton donor toGlu4. On the other hand, N-d1 may also be a protonated, but itdepends if O-g1(Thr36) acts as acceptor. Despite the small

space between O-g1(Thr36) and Nd1(His6), it is available to adda proton on the N-d1(His6) atom by a free rotation aroundCb-Og1 in Thr36 of the oxidized protein. Both the protonatednitrogen atom of His6 is consistent with the peptide bond flip.The geometrical changes at the copper center have been accom-panied by both the peptide bond flip and the loss of watermolecule. Because the metal site consists of the copper ion andthree strong ligand atoms (two histidines and a cysteine), thewater molecule may exist as a hydronium ion to compensatethe excess minus charge at the copper site in the oxidizedpseudoazurin. The loss of water molecule may occur for thereduction of copper plus charge. This may be the direct triggerof the concerted movement around His6. However, it may besaid that both a loss of water molecule and a protonation at theremote histidine may need for the peptide bond flip, becausethe peptide bond flip was found only at pH 7.0 in the pseudoa-zurin from A. faecalis.

The rearrangement of hydrogen bonding interactions of theuncoordinated His6 are observed in A. cycloclastes pseudoa-zurin having Thr36, but not in A. faecalis pseudoazurin withVal36. An important difference between these pseudoazurins isthat Thr36 fixes the position of the imidazole ring of His6 byforming the strong hydrogen bond to its N-d1 atom. On theother hand, Val36 could not fix the nitrogen atom of His6 andthe hydrogen bond between N-e2(His6) and O-e1(Glu4) wasalready formed in the oxidized state. This is why a smallpeptide bond flip was observed in A. faecalis pseudoazurin atpH 7.0 (24).

The protonated pseudoazurin at the His6 position indicated arelatively higher redox potential (42). The protonation at His6

is important to reduction of the protein, and then, the peptidebond flip occurs in the concomitant region from Ile34 to Thr36

upon reduction. Since the His6 residue is not so far from thecopper site (12 Å), this fact might be associated with the theelectron transfer mechanism.

Acknowledgments—We are grateful for helpful and stimulating dis-cussions of Dr. Chris Dennison, University College Dublin. We are alsograteful to Professor N. Sakabe, Dr. N. Watanabe, Dr. Suzuki, and Dr.Igarashi for support in data collection at KEK, Japan.

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Kohzuma and Yasushi KaiTsuyoshi Inoue, Nobuya Nishio, Shinnichiro Suzuki, Kunishige Kataoka, Takamitsu

REMOTE HISTIDINEAREDOX FORMS WITH THE REARRANGEMENT OF HYDROGEN BOND AT

: CONCERTED MOVEMENT OF COPPER SITE INAchromobacter cycloclastesCrystal Structure Determinations of Oxidized and Reduced Pseudoazurins from

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