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
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ABSTRACT |
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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- 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 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 Small inorganic complexes such as
[Fe(CN)6]3 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- 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 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 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 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.
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- 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- 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- 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- 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.
1(His6) and O-
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-
2(His6)
and O-
1(Glu4) is formed with a distance of 3.03 Å,
while the hydrogen bond between
N-
1(His6)-O-
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
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.
-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
-helices at the C
terminus, whereas azurin has an
-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).
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.
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 115.0°,
= 60.0°,
and
= 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
= 115.40°,
= 62.23°, and
= 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(2
i
calc) and
(Fo
Fc)exp(2
i
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
Data collection and final refinement statistics for the oxidized and
reduced pseudoazurins from Ach. cycloclastes
-strands, forming two
-sheets, and two C-terminal
-helices.
-Sheet I consists of
four
-strands: S1, residues 2-8; S2a, 17-19; S3, 30-34; S6,
64-67, and
-sheet II contains five
-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
-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).
View larger version (36K):
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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).
View larger version (38K):
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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.
Metal coordinations of pseudoazurins
1(His6) forms a
hydrogen bond to O-
1(Thr36) with a distance of 2.73 Å in the oxidized pseudoazurin. Both the O-
1(Thr36) and
the N-
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-
2 of His6 to O-
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 C
and
C
atoms of Pro35 have moved by 0.93 and 2.16 Å, respectively.
View larger version (150K):
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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- 1(His6)-O-
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-
1(Thr36). On
the contrary, the distance of
O-
2(Glu4)-N-
2(His6) is changed from 3.47 Å to 3.07 Å and a new hydrogen bonding is formed.
C-
(Pro35) and C-
(Pro35) moved 0.93 Å and
2.16 Å, respectively, and the dihedral angle of
C
-C
and C
-C
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).
Change of main chain conformational angles
2(His6)-O-
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-
1(Thr36) atom and the N-
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.
View larger version (28K):
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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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-
1 may also be a protonated, but it depends if
O-
1(Thr36) acts as acceptor. Despite the small space
between O-
1(Thr36) and N
1(His6), it is
available to add a proton on the N-
1(His6) atom by a
free rotation around C
-O
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.
1 atom. On the other hand,
Val36 could not fix the nitrogen atom of His6
and the hydrogen bond between N-
2(His6) and
O-
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).
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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The abbreviation used is: r.m.s., root mean square.
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