The Structure and Unusual pH Dependence of Plastocyanin from
the Fern Dryopteris crassirhizoma
THE PROTONATION OF AN ACTIVE SITE HISTIDINE IS HINDERED BY
-
INTERACTIONS*
Takamitsu
Kohzuma
§,
Tsuyoshi
Inoue¶,
Fuminori
Yoshizaki
,
Yuki
Sasakawa
,
Kazuhiko
Onodera
,
Shigenori
Nagatomo**,
Teizo
Kitagawa**,
Sachiko
Uzawa
,
Yoshiaki
Isobe
,
Yasutomo
Sugimura
,
Masaharu
Gotowda¶, and
Yasushi
Kai¶
From the
Faculty of Science, Ibaraki University,
Mito, Ibaraki 310-8512, Japan, the ¶ Graduate School of
Engineering, Osaka University, Suita, Osaka 565-0871, Japan, the
Faculty of Science, Toho University, Funabashi, Chiba 274-8510, Japan, and the ** Institute for Molecular Science, Okazaki,
Aichi 444-8585, Japan
 |
ABSTRACT |
Spectroscopic properties, amino acid sequence,
electron transfer kinetics, and crystal structures of the oxidized (at
1.7 Å resolution) and reduced form (at 1.8 Å resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are
presented. Kinetic studies show that the reduced form of
Dryopteris plastocyanin remains redox-active at low pH,
under conditions where the oxidation of the reduced form of other
plastocyanins is inhibited by the protonation of a solvent-exposed
active site residue, His87 (equivalent to His90
in Dryopteris plastocyanin). The x-ray crystal structure
analysis of Dryopteris plastocyanin reveals
-
stacking between Phe12 and His90, suggesting
that the active site is uniquely protected against inactivation. Like
higher plant plastocyanins, Dryopteris plastocyanin has an
acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is
smaller. In the reactions of Dryopteris plastocyanin with
inorganic redox reagents, the acidic patch (the "remote" site) and
the hydrophobic patch surrounding His90 (the "adjacent"
site) are equally efficient for electron transfer. These results
indicate the significance of the lack of protonation at the active site
of Dryopteris plastocyanin, the equivalence of the two
electron transfer sites in this protein, and a possibility of obtaining
a novel insight into the photosynthetic electron transfer system of the
first vascular plant fern, including its molecular evolutionary
aspects. This is the first report on the characterization of
plastocyanin and the first three-dimensional protein structure from
fern plant.
 |
INTRODUCTION |
Plastocyanin is an electron transfer protein having a single
copper atom at the active site. Plastocyanin functions as an electron
carrier protein between the cytochrome
b6f complex and P700+ in
oxygenic photosynthesis (1). Plastocyanin indicates intense absorption
band due to the SCys
Cu(II) charge transfer at visible region and a narrow hyperfine coupling constant in the EPR spectra of
the oxidized form (2). The electronic structure of plastocyanin has
been reported by several groups (3-5). Solomon and co-workers (6) gave
an implication for the electron transfer reaction mechanisms on the
basis of the electronic structure of plastocyanin. The Cu(II)
half-occupied highest occupied molecular orbital is the redox active
orbital in blue copper proteins and plays a key role in the electron
transfer reaction.
Plastocyanin consists of 97-105 amino acid residues. The x-ray
crystallographic structures of poplar (7-9), Enteromorpha prolifera (10), and Chlamydomonas reinhardtii (11)
plastocyanins have been determined, and the solution structures of the
proteins from Anabaena variabilis (12), parsley (13), French
bean (14), and Scenedesmus obliquus (15) have been given by
NMR spectroscopy. The copper atom is located at the loop region of
eight-stranded
-barrel structure and beneath the protein surface at
a depth of 5-10 Å, with two histidines, one cysteine, and one
methionine as ligand groups.
Two regions on the surface of the plastocyanin molecule have been
discussed as potential binding sites for electron transfer partners: an
acidic patch (the "remote" site) and a hydrophobic patch (the
"adjacent" site). Small inorganic complexes such as [Fe(CN)6]3
and
[Co(phen)3]3+ (where phen indicates
1,10-phenanthroline)1 have
been used as redox probes. A great deal of evidence from kinetics
measurements suggests that the adjacent and remote sites are the sites
of electron transfer to [Fe(CN)6]3
and
[Co(phen)3]3+, respectively (16). In higher
plant plastocyanins, the adjacent site consists of conserved
hydrophobic amino acid residues surrounding the solvent-exposed
His87, and the remote site is an acidic patch comprising
Asp42, Glu43, Asp44,
Glu59, Glu60, Asp61, and
Glu68 as well as the invariant residue Tyr83
(9). Electron transfer reaction studies of the nitrated plastocyanin at
the remote site Tyr83 indicated the possible interaction
with cytochrome f at the site of Tyr83 (17).
Site-directed mutagenesis studies of pea plastocyanin has demonstrated
that the solvent-exposed Tyr83 is essential for binding and
electron transfer reactions (18). From these reaction studies and
comparative investigations using the acidic patch mutants, it has been
suggested that a physiological electron donor, cytochrome f,
might interact with the acidic patch of plastocyanin. Qin and Kostic
(19, 20) have suggested that the rearrangement of the electron transfer
site between plastocyanin and cytochrome c (as a
model of cytochrome f) causes the gating of the
electron transfer. Very recently, computational simulation also
indicates that the rearrangement is optimized through the
-cation
interaction between the phenol moiety of the remote electron transfer
site Tyr83 and the
-amino group of lysine side chain
of cytochrome f to the effective electron transfer
interaction mode (21-23). Recent solution structure analysis of
spinach plastocyanin indicates that the complex formation with
cytochrome f dominantly occurs through the hydrophobic
moiety of plastocyanin (24).
Plastocyanin is distributed among wide variety of oxygenic
photosynthetic organisms involving cyanobacteria, green algae, and
higher plants. The expression level of plastocyanin in a part of
cyanobacteria and green algae is regulated by the levels of copper
concentration in the culture medium (25). Although the presence of
plastocyanin in species of fern has been recognized (26), no
plastocyanin from a fern has been characterized previously. A novel
plastocyanin has now been isolated and purified from the fern
Dryopteris crassirhizoma.
We here report the isolation, spectroscopic properties, amino
acid sequence, electron transfer kinetics, and crystal structures of
the oxidized (at 1.7 Å resolution) and reduced form (at 1.8 Å resolution) of a novel plastocyanin from the fern D. crassirhizoma. This is the first report on the characterization of
plastocyanin from ferns and also the first three-dimensional protein
structure from ferns.
 |
EXPERIMENTAL PROCEDURES |
Isolation and Purification of Plastocyanin--
Leaves of
D. crassirhizoma Nakai, collected on a mountain in Nagano
Prefecture, Japan, were homogenized in 0.2 M potassium phosphate buffer (pH 8.0) with an automatic mortar. The homogenate was
centrifuged to remove debris and subjected to ammonium sulfate fractionation (0.3-1.0 saturation), ion exchange column chromatography with DEAE-cellulose and DEAE-Sephacel (Amersham Pharmacia Biotech), hydrophobic chromatography with Butyl-Toyopearl 650 M
(Tosoh), and gel filtration with Bio-Gel P-10 (Bio-Rad). Protein
homogeneity was checked with analytical SDS-polyacrylamide gel electrophoresis.
Amino Acid Sequence Determination--
The complete amino acid
sequence of Dryopteris plastocyanin was determined as
described (27), except that carboxymethyl-plastocyanin was subjected to
lysyl endopeptidase digestion (molar ratio of enzyme/substrate = 1/400, in 0.01 M Tris-HCl buffer, pH 8.6, at 37 °C for
6 h) or cyanogen bromide cleavage (molar ratio of
chemical/protein = 500, in 70% formic acid at room temperature
for 20 h), and an Applied Biosystems 473A protein sequencer was
used. The sequences of plastocyanin and peptides isolated were
confirmed by amino acid analysis.
Sequence Alignment--
Amino acid sequence data were obtained
from the data base SWISS-PROT release 36 (28), and each sequence was
aligned by using CLUSTAL X version 1.64b (29). The putative processing
sites are those shown in the data base or the reference cited therein.
Spectroscopic Measurements--
UV-visible spectra were
determined on a Beckman DU-7500 recording spectrophotometer. Resonance
Raman spectra were measured at room temperature with a spinning cell
(1800 rpm; 5-mm diameter), and Raman shifts were calibrated to an
accuracy of 1 cm
1 using indene and carbon tetrachloride.
Resonance Raman scattering was excited at 607 nm by a rhodamine 6G dye
laser (Spectra Physics, model 375B) pumped by an Ar+ ion
laser (Spectra Physics, model 2017) and an Astromed CCD 3200 detector
attached to a single monochromator (Ritsu Oyo Kogaku, DG-1000). The
laser power was adjusted to 60 mW at the sampling point.
Kinetic Measurements--
All reactions were monitored on an
Otsuka Electronics (Osaka, Japan) Photal RA-401 stopped flow
spectrophotometer. Electron transfer reactions of plastocyanin with
inorganic complexes were monitored at 590 nm at 25 °C. All kinetic
parameters were calculated using the program IgorPro
(WaveMetrics, Lake Oswego, OR). The kinetic data were analyzed using
the following equation (16).
|
(Eq. 1)
|
where the acid dissociation constant Ka and
the rate constants are as defined in Equations 2-4.
|
(Eq. 2)
|
|
(Eq. 3)
|
|
(Eq. 4)
|
Electrochemical Measurements--
Cyclic voltammetry
was carried out using a BAS model CV-27 Voltammograph (Bioanalytical
Systems Inc.). Modification of gold electrode was described in the
previous report (30). 2,2'-Diethylaminoethanethiol (Aldrich) was used
for the modification of the electrode as a promoter. A
single-compartment electrochemical cell was used with an Ag/AgCl
reference electrode (Bioanalytical Systems Inc.), and a platinum wire
counter electrode was separated by a vicor glass tip from the working
solution. The electrode potential was calibrated with the potential of
[Co(phen)3]2+/3+ couple. Oxygen was removed
from the working compartment by passing humidified O2-free
argon through the electrochemical cell for 15 min.
X-ray Crystallography--
The hanging drop vapor diffusion
method was applied for the crystallization of plastocyanin using
ammonium sulfate as the precipitant. A hanging drop comprising a 6-µl
droplet of a 10 mg/ml protein solution in 0.1 M sodium
acetate buffer (pH 4.5) and 32% saturated ammonium sulfate was
suspended above 500 µl of a reservoir solution comprising the same
buffer, 1 mM sodium azide and 64-65% saturated ammonium
sulfate. Single crystals with maximum dimensions 0.4 × 0.4 × 0.2 mm3 were obtained. All processes were
carried out at 20 °C. The reduced form was prepared by soaking a
crystal in the mother liquid containing 10 mM sodium
ascorbate. The characteristic blue color disappeared after 10 min.
Intensity data for both the oxidized and reduced forms were collected
from single crystals on a Rigaku RAXIS-IIc imaging plate, using
CuK
radiation from a rotating anode x-ray generator,
Rigaku RU-300 with fine-focused beam and
-filtered (40 kV, 100 mA).
The unit cell was determined to be hexagonal with a = b = 73.15 Å and c = 31.10 Å by
autoindexing software on the RAXIS. The data were reduced in Laue group
6/m, and reflections with (I/
) > 1 were accepted. The asymmetric
unit in space group P61 includes one
plastocyanin molecule (molecular mass = 10,000 dalton) with a
Vm value of 2.4 Å3/dalton (31).
From the value of Vm the estimated solvent
content is 49%. Intensity data sets were initially collected up to 2.0 Å resolution. A second intensity data set for reduced form was collected up to 1.8 Å resolution. Among 28,202 accepted observations up to 1.8 Å resolution, 8,478 independent reflections were obtained with an Rmerge of 6.1% and a completeness of
93.5%. A second set of intensity data for the oxidized form was
collected at
= 1.00Å with synchrotron radiation at the Photon
Factory using Sakabe's Weisenberg camera for macromolecules (32).
Among 69,003 accepted observations up to 1.7 Å resolution, 10,220 independent reflections were obtained with an
Rmerge of 8.9% and a completeness of 96%. The
crystal structure of oxidized form was solved by the molecular replacement method with the program of AMoRe (33) in
CCP4 program package (34). The molecular structure of
plastocyanin from poplar (35) was used as the starting model. Model
rebuilding was performed with the program FRODO (35).
Refinement of the oxidized structure was carried out at 1.7 Å resolution by the simulated annealing refinement method
(X-PLOR (34)). Water molecules were added in three steps by
using the WATPEAK program in CCP4 (34). The current structure of oxidized plastocyanin includes 758 protein atoms
(nonhydrogen), one metal ion and 47 water molecules. After rebuilding
and several cycles of refinement, the R-factor and free
R-factor are 24.1 and 28.8% for 9,943 unique reflections in
the range of 6.0 to 1.7Å. The structure of reduced plastocyanin including 38 water molecules is refined up to 1.8 Å resolution with an
R-factor and a free R-factor of 20.7 and 22.6%, respectively.
 |
RESULTS AND DISCUSSION |
Plastocynain from D. crassirhizoma consists of 102 amino acid residues. The complete amino acid sequence of the
plastocyanin has been determined (Fig.
1). The amino acid sequence of D. crassirhizoma plastocyanin is much different from those of the
seed plant proteins so far reported (Fig.
2). Percentage divergences between the
fern sequence and each of the seed plant sequences are 62-67%; in
contrast, those among the seed plant sequences are 2-37%. Because the
sequence for D. crassirhizoma is identical to that for
another fern, Polystichum longifrons, except for 4 amino
acid residues,2 fern
plastocyanin sequences might be similar to each other.

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Fig. 1.
Amino acid sequence of Dryopteris
plastocyanin. K- and B- refer to the
peptides derived by lysyl endopeptidase digestion and by cyanogen
bromide cleavage, respectively. Arrows indicate amino acid
residues identified by automated Edman degradation; dotted
arrows show ambiguous identifications.
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Fig. 2.
Sequence alignment of plastocyanins from a
fern and seed plants. Abbreviations of plant names and the
accession numbers of SWISS-PROT (in parenthesis) are as follows:
Drycr, D. crassirhizoma (this work);
Dauca, Daucus carota (P20422); Petcr
a, Petroselinum crispum plastocyanin a
(P17341); Horvu, Hordeum vulgare (P08248);
Orysa, Oryza sativa (P20423); Arath,
Arabidopsis thaliana (P11490); Capbu,
Capsella bursa-pastoris (P00294); Cucpe,
Cucurbita pepo (P00292); Cucsa, Cucumis
sativus (P00293); Merpe, Mercurialis
perennis (P00295); Samni, Sambucus nigra
(P00291); Spiol, Spinacia oleracea (P00289);
Lacsa, Lactuca sativa (P00290); Silpr,
Silene pratensis (P07030); Phavu, Phaseolus
vulgaris (P00287); Lyces, Lycopersicon
esculentum (P17340); Soltu, Solanum
tuberosum (P00296); Nicta a',
Nicotiana tabacum plastocyanin a' (P35476);
Nicta b', Nicotiana tabacum
plastocyanin b' (P35477); Solcr, Solanum
crispum (P00297); Rumob, Rumex obtusifolius
(P00298); Pissa, Pisum sativum (P16002);
Vicfa, Vicia faba (P00288); Popni a,
Populus nigra plastocyanin a (P00299);
Popni b, Populus nigra plastocyanin b
(P11970). Acidic amino acids are shown in red; basic amino
acids are in blue. The conserved amino acids are marked by
asterisks.
|
|
Dryopteris plastocyanin has a significantly different
electronic absorption spectrum from the usual higher plant
plastocyanins (Fig. 3). The electronic
absorption spectrum of Dryopteris plastocyanin has maxima at
463 nm (
= 1000 cm
1 M
1), 590 nm (
= 4700 cm
1 M
1), and 753 nm (
= 2000 cm
1 M
1), and a
shoulder peak at 410 nm (
= 900 cm
1
M
1) in the visible region. The absorption
band at 590 nm has been assigned to the Cys(Sp
)
Cu2+ (2, 3). In comparison with the spectra of higher plant plastocyanins, the most intense absorption band at 590 nm is shifted to
a shorter wavelength by 7 nm, and the intensity of the absorption band
at 463 nm is higher. This might reflect a different electronic structure of the active site. Fig. 4
shows a resonance Raman spectrum of Dryopteris plastocyanin
obtained by excitation at 590 nm. Resonance Raman spectra of blue
copper proteins including plastocyanin excited in resonance with the
SCys
Cu2+ charge transfer
band characteristically have one or two Raman bands in the 250 cm
1 region and multiple Raman bands in the 330-490
cm
1 region. The former and latter are associated with
Cu-NHis and Cu-SCys moieties, respectively (36,
37). Very recently, the lower frequency band at 267 cm
1
has been assigned to the Cu-NHis in poplar plastocyanin by
Dong and Spiro (38). Dryopteris plastocyanin has a unique
spectrum in the Cu-SCys region, with two strong peaks at
426 and 381 cm
1, and additional small peaks at 491, 447, 410, 392, 367, and 339 cm
1. According to the assignment
in poplar plastocyanin (38), the 426 and 381 cm
1 bands
may arise from the coupling of the modes of
(Cu-SCys),
(C
C
N), and
(C
C
S), and remains are also assignable to internal modes of Cys residue and/or the coupling with
(Cu-SCys) (38). There is also an isolated
Cu-NHis mode close to 262 cm
1. This might be
contributed from the stretching between copper and coordinated
imidazole nitrogen atom of solvent exposed His90. The
overall similarity of the resonance Raman spectrum to those of blue
copper proteins suggests that the coordination at the copper site of
Dryopteris plastocyanin is generally similar to that
in higher plant plastocyanins (38, 39).

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Fig. 3.
UV-visible absorption spectrum of
Dryopteris plastocyanin (100 µM) at pH 7.0 (20 mM
phosphate, 0.1 M NaCl).
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Fig. 4.
Resonance Raman spectrum of
Dryopteris plastocyanin (1 mM) at pH 7.0 (20 mM phosphate, 0.1 M NaCl).
|
|
Two regions on the surface of the plastocyanin molecule have been
discussed as potential binding sites for electron transfer partners: an
acidic patch (the remote site) and a hydrophobic patch (the adjacent
site). A great deal of evidence from kinetics measurements suggests
that the adjacent and remote sites are the sites of electron transfer
to [Fe(CN)6]3
and
[Co(phen)3]3+, respectively (16). The
calculated second-order rate constants (pH 7.5) for the oxidation of
Dryopteris plastocyanin by
[Fe(CN)6]3
and
[Co(phen)3]3+ are 1.87 (± 0.02) × 105 and 1.69 (± 0.04) × 103
M
1 s
1, respectively. The
electron transfer rate constants for the reaction with
[Fe(CN)6]3
and
[Co(phen)3]3+ seem to be almost identical to
the values for higher plant plastocyanins. The crystal structure of
Dryopteris plastocyanin has been determined at a resolution
of 1.7 Å (oxidized) and 1.8 Å (reduced). The root mean square
difference between the backbone structures of poplar and
Dryopteris plastocyanins is 0.74 Å. The structure of
Dryopteris plastocyanin demonstrates the migration of the
acidic patch (but not the invariant Tyr86) toward the
adjacent site (Fig. 5). The disappearance
of the acidic residues from the remote site reduces the electrostatic repulsion for negatively charged
[Fe(CN)6]3
, thus facilitating electron
transfer to [Fe(CN)6]3
at the remote site.
The electron transfer rate constants, similar to those for higher plant
plastocyanins (16) despite the significant structural changes, support
the hypothesis that the remote site including Tyr86
(corresponding to Tyr83 for usual higher plants) and the
adjacent site including His90 (His87 for usual
higher plants) are the electron transfer site with [Fe(CN)6]3
and
[Co(phen)3]3+, respectively. The kinetic
results also indicate that the remote and adjacent sites in
Dryopteris plastocyanin are equally efficient for electron
transfer, as predicted by the electronic structure analysis (3,
40).

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Fig. 5.
Schematic representation of the structure of
Dryopteris plastocyanin (46).
Dryopteris plastocyanin indicates three-turned -helix
structure constituted of the 10 amino acid residues (positions 49-58)
Gly, Thr, Val, Ala, Ser, Glu, Leu, Lys, Ala, and Ala.
|
|
The pH dependence of the rate constants for the reactions of various
plastocyanins with [Fe(CN)6]3
and
[Co(phen)3]3+ complexes have been reviewed
(16). The variation with pH of the second-order rate constants
(ksec) for the oxidation of
Dryopteris plastocyanin by
[Fe(CN)6]3
and
[Co(phen)3]3+ complexes is illustrated in
Fig. 6. The acid dissociation constants, pKa, associated with oxidation by
[Fe(CN)6]3
and
[Co(phen)3]3+ are 5.9 ± 0.1 and
6.2 ± 0.3, respectively. Studies of the electron transfer
kinetics and crystal structure of plastocyanin as functions of pH have
suggested that electron transfer is inhibited at low pH by the
protonation of the active site His87 (16). In contrast,
Dryopteris plastocyanin does not become redox-inactive under
acidic conditions. This suggests that in Dryopteris
plastocyanin the active site His90 (His87 in
higher plants) does not become protonated. In the structure of
Dryopteris plastocyanin, the imidazole ring of the
coordinated His90 is stacked at a van der Waals' contact
distance against the phenyl group of Phe12 (Fig.
7). We suggest that the
-
stacking
interaction stabilizes the Cu-N(His90) bond, inhibits the
rotation of the His90 imidazole ring, and thus prevents the
imidazole group from becoming protonated at acidic pH.

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Fig. 6.
The dependence of second-order rate constants
on pH for the oxidation of reduced Dryopteris
plastocyanin with [Fe(CN)6]3
(filled circles) and
[Co(phen)3]3+ (open
circles). Reactions of plastocyanin with inorganic
complexes were monitored at 590 nm at 25 °C. Calculated kinetic
parameters were: [Fe(CN)6]3 ,
k0 = 1.97 (± 0.04) × 105
M 1 s 1,
kH = 2.5 (± 0.8) × 105
M 1 s 1, pKa = 5.9 ± 0.1; [Co(phen)3]3+,
k0 = 2.01 (± 0.07) × 103
M 1 s 1,
kH = 890 ± 150 M 1 s 1, pKa = 6.2 ± 0.3.
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Fig. 7.
The front (A) and side
(B) views of the active site represented by
ball-and-stick model (47). The aromatic rings of Phe12
and His90 (His87 for poplar) are stacked with
the distance of 3.5 Å.
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The structure of the reduced protein indicates only 0.15 Å root mean
square deviation from the oxidized conformation. The bonding parameters
for the oxidized (pH 4.5) and reduced (pH 4.5) forms strongly support
the lack of protonation at the active site of Dryopteris
plastocyanin in contrast to higher plant plastocyanins where
significant structural changes are caused by the dissociation of
His87 (His90 for Dryopteris) from
the copper atom (Table I). A stacking
interaction between the coordinated imidazole ligand of histamine and
the phenyl group of phenylalanine was demonstrated in the previous studies of the stability and structure of copper complexes (41). Yamauchi and co-workers (42) have pointed out that the stacking interaction induces stronger imidazole-copper bonding due to the delocalization of electron density on the copper center. By the comparison of electronic absorption spectra of usual seed plant plastocyanins, the electronic absorption spectrum of
Dryopteris plastocyanin indicates the 7 nm blue-shifted
charge transfer band, and an additional absorption band at 410 nm is
recognized in the electronic absorption spectrum (see above). The
resonance Raman excitation profile of poplar plastocyanin has suggested
that the charge transfer band from His
1 to
Cu2+ should be lying into the most intense absorption band
around at 600 nm (38). On the other hand, the charge transfer band between stacked Phe12 and His90 residues would
be expected around 300-400 nm region (42). The differences of
electronic absorption spectra between Dryopteris and usual
higher plant plastocyanins may reflect the stacking structure on the
active site electronic structures.
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Table I
Dimensions of the copper site in oxidized and reduced Dryopteris
plastocyanin at pH 4.5, compared with poplar plastocyanin (9)
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Cyclic voltammetry was performed for Dryopteris plastocyanin
in the potential range +600-0 mV versus normal hydrogen
electrode, and showed a well defined quasi-reversible Faradaic response
with a peak-to-peak separation
Ep of 95mV at
a diethylaminoethanethiol modified gold (DEAET/Au) electrode at pH 7.0 (data not shown). The redox potential of Dryopteris
plastocyanin has been determined to be 387 mV versus normal
hydrogen electrode at pH 7.0. This value is approximately 20 mV higher
than the values for most other plastocyanins (360-370 mV
versus normal hydrogen electrode), although it was expected
that a lower redox potential due to negatively charged acidic amino
acid residues surrounding the hydrophobic part of Dryopteris
plastocyanin would be observed as a decrease in the redox potential in
the mutant plastocyanins (43) and azurin (44) substituted with acidic
amino residues near the copper center. Despite the several acidic amino
acid residues located near the copper center (adjacent site) in the
case of Dryopteris plastocyanin, the redox potential appears
at a higher potential. The remarkable positive value of the redox
potential may reflect the reduction of electron density on the copper
center through the
-
stacking interaction between the coordinated
imidazole ligand of His90 and the phenyl group of
Phe12. The pH dependence of the reduction potential of the
protein displays at least one acid-base equilibrium (data not shown). At the lower pH values the behavior is different from that observed for
other plastocyanins (16, 45) and pseudoazurin (30), which indicate
protonation of solvent-exposed histidine residue located at the active
center. In the usual higher plant plastocyanins, the differences of
redox poteintial,
E between pH 5 and pH 7 have been evaluated to be
50-60 mV, but the corresponding
E value of Dryopteris
plastocyanin has been estimated to be only 18 mV. It is most likely
that the independence of the redox potential on pH also reflects the
stacking interaction between Phe12 and His90
preventing the protonation of the active site His90.
Sykes suggested that the pKa values obtained from
the kinetic experiments using [Co(phen)3]3+
are due to contributions arising from both the acidic patch and the
active site protonation (16). The acid dissociation constant, pKa = 6.2 ± 0.3, obtained from the kinetic
studies of Dryopteris plastocyanin would reflect the pure
protonation process at the acidic patch, because the redox reaction
proceeds without active site protonation. The affinity of cationic
[Co(phen)3]3+ for Dryopteris
plastocyanin should be decreased by the partial neutralization of the
negative charge on the protein molecule, which may explain the lowering
of the activity at low pH (Fig. 6).
These results indicate the significance of the lack of protonation at
the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer
system of the first vascular plant fern, including its molecular
evolutionary aspects.
 |
ACKNOWLEDGEMENTS |
We are grateful to Prof. Hans C. Freeman and
Dr. Panos Kyritsis for careful reading of the manuscript and valuable
suggestions and are also thankful for the helpful and stimulating
discussions of Profs. A. Geoff Sykes, J. Mitchell Guss, Nenad M. Kostic, Osamu Yamauchi, and Klaus Bernauer. We are also grateful to the
Sakabe project of the TARA and Professor Norio Sahashi for identifying D. crassirhizoma.
 |
FOOTNOTES |
*
This work was supported by Grants-in-Aid for Science
Research on Priority Areas from the Ministry of Education, Science,
Sports and Culture, Japan, by the Joint Studies of Program (1997) of the Institute for Molecular Science, and by the Grant-in-Aid for the
Ground Experiment for the Space Utilization from the Japan Space Forum
and National Space Developments Agency of Japan to Kohzuma.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 1KDJ and
1KDI) have been deposited in the Protein Data Bank, Brookhaven National
Laboratory, Upton, NY.
§
To whom correspondence should be addressed: Faculty of Science,
Ibaraki University, Mito, Ibaraki 310-8512, Japan. Tel. and Fax:
81-29-228-8372; E-mail: kohzuma{at}biomol.sci.ibaraki.ac.jp.
2
Y. Nagai and F. Yoshizaki, unpublished results.
 |
ABBREVIATIONS |
The abbreviation used is:
phen, 1,10-phenanthroline.
 |
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