(Received for publication, February 6, 1995; and in revised form, July 11, 1995)
From the
The single type 1 copper protein pseudoazurin from Achromobacter cycloclastes gives reversible electrochemical
behavior at a (4-pyridyl)disulfide-modified gold electrode.
Measurements carried out at 25.0 °C indicate a midpoint reduction
potential of E = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation
of
E
= 59 mV. The diffusion
coefficient and heterogeneous electron transfer rate constant are
estimated to be 2.23
10
cm
s
and 3.7
10
cm
s
, respectively. Also, controlled potential
electrolysis indicates a 1-electron transfer process and a formal
reduction potential of 259 mV versus normal hydrogen electrode
for the Cu(II)/Cu(I) couple. The heterogeneous electron transfer rate
constant determined at the (4-pyridyl)disulfide-modified gold electrode
at pH 4.6 is 6.7
10
cm s
,
consistent with a slower process at the positively charged electrode
surface. At pH 11.3, UV-visible, EPR, and resonance Raman spectra
indicate a conversion of the distorted tetrahedral copper geometry to a
trigonal structure. The trigonal form has elongated axial bonding and
an axial EPR spectrum. At pH 11.3, the reduction potential is further
decreased, and Cu-S bands in resonance Raman spectra at
330-460 cm
are shifted to higher energy
(
10 cm
), consistent with a stronger Cu-S
bond.
The type 1 blue copper proteins are a family of electron
transfer proteins that have a single copper atom at their active site,
an intense absorption band due to the S(Cys) Cu(II) charge
transfer, and a narrow hyperfine coupling constant in the EPR spectra
of the oxidized protein(1) . The copper atom is located beneath
the protein surface at a depth of 5-10 Å(2) , with
two histidines (imidazole (N)), one cysteine (thiolate
(RS
)), and one methionine (thioether (S)) as ligands.
Recent kinetic (3) and theoretical (4, 5) studies on the blue copper protein plastocyanin
have indicated the presence of two distinct electron transfer sites:
(i) the adjacent site at the surface-exposed histidine of the
hydrophobic patch
6 Å from the copper and (ii) the remote
site involving acidic patch region
15 Å from the copper with
acidic residues on either side of the exposed Tyr-83. Corresponding
sites appear to be effective for electron transfer in the
plastocyanin-like domain of ascorbate oxidase (and presumably other
copper oxidases) and are considered here for the single type 1 copper
protein pseudoazurin.
Pseudoazurin (14 kDa) is known to be a
component of at least four bacteria, Achromobacter
cycloclastes(6) , Pseudomonas AM1(7) , Alcaligenes faecalis(8) , and Thiosphaera
pontotropha(9) , where it functions as an electron carrier
in the respiratory chain of the microorganism. The A.
cycloclastes(10) , A. faecalis(8) , and T. pontotropha(9) pseudoazurins are electron donors
to their respective nitrite reductases. A. cycloclastes pseudoazurin, which has a pI of 8.4 and a charge (from the amino
acid composition, with Asp and Glu as 1
and Arg and
Lys as 1
) of 1
at pH 7.0, is the
subject of this study. The three-dimensional x-ray structure of A.
faecalis pseudoazurin has been reported(11, 12) ,
and it has been confirmed that the copper ion is coordinated to two
histidines (His-40 and His-81), one cysteine (Cys-78), and one
methionine (Met-86) in a distorted tetrahedral configuration. The
Cu-S(Met-86) distance (2.76 Å) is shorter than that observed in
the plastocyanins from poplar leaves (2.83 Å) (13) and Enteromorpha prolifera (2.93 Å) (14) as well as
in azurin from Alcaligenes denitrificans (3.11
Å)(15) . Pseudoazurin has a distorted tetrahedral copper
center similar to that of plastocyanin, in contrast to azurin, which
has a distorted trigonal copper center. More recently, the x-ray
structure of pseudoazurin from A. cycloclastes has been
reported (Fig. 1)(16) , and the structure is almost
identical to that of pseudoazurin from A.
faecalis(11, 12) and Pseudomonas AM1(17) .
Figure 1: Structure of pseudoazurin from A. cycloclastes (47) drawn by the program MOLSCRIPT(48) .
A single compartment electrochemical cell
was used with an Ag/AgCl reference electrode (Bioanalytical Systems,
Inc.) and a platinum wire counter electrode separated by a Vicor glass
tip from the working solution. Controlled potential electrolysis of
pseudoazurin was investigated with a Bioanalytical Systems Model CV-27
voltammograph. Electronic absorption spectra were monitored during the
course of electrolysis using a Shimadzu spectroscopic flow cell. Oxygen
was removed from the working compartment by passing humidified
O-free argon through the electrochemical cell for 15 min.
Cyclic voltammograms were obtained for pseudoazurin in the
potential range +600 to 0 mV versus NHE and
showed a well defined quasi-reversible faradaic response, with a
midpoint potential of E
= 260 mV versus NHE and a peak-to-peak separation of
E
= 59 mV at a 4-pyds/Au electrode at
pH 7.0 (Fig. 2). The cathodic peak current (i
Figure 2: Cyclic voltammogram of pseudoazurin (100 µM) from A. cycloclastes at a scan rate of 10 mV/s (0.1 M phosphate buffer, pH 7.0) and at 25 °C.
Figure 3:
A,
plot of the observed current, i =
0.4463nF
R
T
AC
D
v
,
as a function of potential scan rate, where n = number
of electrons, F = Faraday constant, R =
gas constant, T = temperature, A = area
of electrode surface (0.0211 cm
), C
= bulk concentration of
substance, D
= diffusion coefficient of
substance, and v = potential scan rate. B,
plot of the linear relationship between the kinetic parameter
= k
/(
DnFv/RT)
and potential scan rate.
Controlled
potential electrolysis of pseudoazurin was also performed at a
4-pyds/Au mesh electrode, and UV-visible spectra were measured
simultaneously at various electrode potentials (Fig. 4A). A decrease in the intense blue absorbance of
pseudoazurin at 593 nm indicates reduction of Cu(II) to Cu(I).
Reoxidation of electroreduced pseudoazurin could be achieved,
confirming that electron exchange of pseudoazurin occurs at the
electrode surface without denaturation of the protein. Spectra observed
at intermediate potentials are a measure of the ratio of oxidized to
reduced forms present. Fig. 4B shows a typical plot of E against
log([pseudoazurin
]/[pseudoazurin
]).
The slope of the straight line is 61 mV, which compares favorably with
the predicted nernstian value of 59 mV for a 1-electron process. At 25
°C and pH 7.0 (0.10 M phosphate), the reduction potential (E`
) of pseudoazurin from the potential axis
intercept is 259.1 ± 0.2 mV versus NHE, which is in
good agreement with the value of 260 mV obtained from cyclic and
square-wave voltammetry.
Figure 4: A, controlled potential electrolysis of pseudoazurin (170 µM) at 25 °C and pH 7.0 (0.1 M phosphate) showing absorption spectra of pseudoazurin during electrolysis at various electrode potentials: 503.8 (spectrum a), 303.8 (spectrum b), 275.3 (spectrum c), 263.1 (spectrum d), 243.8 (spectrum e), 225.2 (spectrum f), 205.3 (spectrum g), and 104.3 (spectrum h) mV versus NHE, respectively. The electrolyzed solution of pseudoazurin at each electrode potential was passed through a spectroscopic flow-cell and recovered into the electrochemical cell. B, the Nernst plot for the controlled potential electrolysis results. pAz, pseudoazurin.
The pH dependence of the reduction
potential of the protein displays at least two acid-base equilibria (Fig. 5). At the lower pH values, the behavior is similar to
that observed for plastocyanin (23, 24, 25) and is attributed to the
acid-base properties of an active-site histidine of the Cu(I) protein.
From the difference between the pK values for
amino acid residues in the two oxidation states of the protein, it is
possible to determine, using , the effect of protonation or
deprotonation of specific amino acid residues on the redox potential of
the protein(26) :
Figure 5: Variation of the reduction potential for the pseudoazurin Cu(II)/Cu(I) couple at different pH values. The data shown by open and closed circles are from cyclic voltammetry and square-wave voltammetry, respectively.
where K and K
are the proton dissociation
constants for the residues i in the reduced and oxidized
proteins, respectively. Then the electrochemical data can be fitted to
two pK
values of 6.6 and 10.4, respectively.
In
the case of plastocyanin, it has been demonstrated that protonation
results in a loss of redox activity(27) . X-ray
crystallographic studies on reduced plastocyanin at different pH values
(3.8-7.8) have confirmed the effect as an
H-induced dissociation of His-87, resulting in the
Cu(I) being coordinated by His-37, Cys-84, and Met-92 in a trigonal
arrangement(28) . Similarly, the increase in reduction
potential of pseudoazurin at the lower pH values is assigned to the
protonation of the corresponding histidine residue (His-81,
pK
= 6.6). At pH 11.3, the reduction
potential of pseudoazurin is less, E
= 201 mV versus NHE, which is assigned here to a
structural transition brought about by the acid dissociation of the
-NH
-amino group of Lys-77
(pK
= 10.4).
A. cycloclastes Cu(II) pseudoazurin exhibits three intense charge transfer bands
() at 452 (
= 1400 M
cm
), 593 (
= 3700), and 753 (
= 1800) nm at pH 7.0. Recent
works suggest that both the bands at around 600 and 450 nm in the
visible absorption spectra of blue copper proteins correspond to S(Cys)
Cu(II) electronic transitions(29, 30) . The
visible spectrum of Cu(II) pseudoazurin is not changed drastically in
the pH range 4.6-10.4. At pH 11.3, the intensity of the peak at
593 nm increased (
= 3900), and that at 452 nm decreased
(
= 1100), indicating a structural transition at the Cu(II) (Fig. 6). Han et al.(29) have pointed out that
the sum of the
values for these two bands is similar for most
type 1 copper proteins. At pH 7.0, the sum of the two
values at
452 and 593 nm for oxidized pseudoazurin is 5100 M
cm
, whereas at pH
11.3, it is 5000 M
cm
.
This constancy of the sum is in agreement with the behavior of other
blue copper proteins and confirms that the changes are not due to
denaturation.
Figure 6: UV-visible absorption spectra of pseudoazurin (100 µM) at pH 7.0 (solid line) and pH 11.3 (broken line).
The EPR spectra of the type 1 blue copper proteins can
be either axial or rhombic. At pH 7.0, the spectrum of pseudoazurin
shows a typical rhombic signal with anisotropic spin hamiltonian
parameters, g = 2.01 (A
= 6.7 milliteslas), g
= 2.09,
and g
= 2.21 (A
= 5.0 milliteslas) (Fig. 7A). The EPR
spectra of pseudoazurin do not change with pH in the range
4.6-10.4, consistent with no active-site effect in the Cu(II)
form of the protein. However, at pH 11.3, the rhombic signal becomes
almost axial with anisotropic EPR parameters, g
= 2.06 and g
= 2.20 (A
= 6.3 milliteslas) (Fig. 7B).
Figure 7: EPR spectra of pseudoazurin (100 µM) at pH 7.0 (0.1 M phosphate) and a microwave frequency of 9.269 GHz (5 milliwatts) (A) and at pH 11.3 (0.1 M phosphate) and a microwave frequency of 9.219 GHz (5 milliwatts) (B) at 77 K. The modulation amplitude was 6.3 G (100 kHz) at all EPR measurements. mT, milliteslas.
Resonance Raman spectra of blue copper
proteins are enhanced by excitation, leading to characteristic spectra
with one or two vibrational modes in the 250-280 cm region and as many as nine vibrational modes in the 330-490
cm
region. The modes at the lower frequencies are
assigned to Cu-His transitions and those at higher frequencies to
Cu-Cys transitions(31) . A. cycloclastes pseudoazurin
has its own unique spectra in the Cu-Cys region, with one strong peak
at 392 cm
and an additional seven smaller peaks at
454, 439, 411, 383, 368, 353, and 335 cm
(Fig. 8A). There is also an isolated Cu-His mode close
to 257 cm
. The multiplicity of vibrational modes
between 330 and 460 cm
has been ascribed to
kinematic coupling of the Cu-S(Cys) stretch with deformations of
both the
(S-C
-C
) and
(C
-C
-N) modes of the
cysteine ligand(32, 33, 34) . The Raman bands
of
(Cu-S) are at 449, 424, 400, 383, 361, and 337
cm
, and at pH 11.3, an isolated Raman band
corresponding to Cu-N(His) is observed at 269 cm
(Fig. 8B). (
)These spectra indicate
that the 439, 411, 392, 353, and 257 cm
Raman bands
have been shifted to 449, 424, 400, 361, and 269 cm
,
respectively. These shifts to higher energy (
10
cm
) indicate that the Cu-S(Cys) and
Cu-N(His) bonds are stronger and shorter than those observed for
pseudoazurin at neutral pH.
Figure 8: Resonance Raman spectra of pseudoazurin (200 µM) at pH 7.0 (0.1 M phosphate) (A) and at pH 11.3 (0.1 M phosphate) (B).
The transition occurring at pH 11.3 can be monitored by
the effect of pH on the visible spectrum of the Cu(II) protein. It is
referred to here as a blue copper active-site transition. The ratio (R) of the
values at 452 and 593 nm is 0.38 at neutral
pH and 0.28 at higher pH values (11.3). The value of R is
believed to reflect the strength of the axial
coordination(29) . The larger R values indicate
stronger axial coordination, leading to a distorted tetrahedral copper
environment as in the type 1 copper site of nitrite reductase from A. cycloclastes (R =
1.22)(29, 30) . The smaller value indicates a weaker
axial binding as in the case of azurin (R = 0.11),
where the Cu(II) coordination is almost trigonal(15) . The EPR
spectrum of pseudoazurin at pH 11.3 also suggests that the structural
transition at pH 11.3 makes the axial coordination weaker and that the
Cu-S(Met-86) bond is elongated compared with the situation at
neutral pH. The axial distortion of the copper center is also supported
from the Raman shifts (
10 cm
) of the
Cu-S(Cys) and Cu-N(His) vibrational modes to higher energy
at pH 11.3. As a result of the axial distortion, the copper atom lies
more in the plane of the three equatorial ligands His-40, His-81, and
Cys-78 (Fig. 9). It is significant that the trigonal to
tetrahedral blue copper transition in the H117G azurin mutant leads to
Cu-S(Cys-112) Raman bands coupled with cysteine deformations that
are 10-40 cm
lower in
energy(35, 36) . A trigonal geometry at the copper,
with the S(Cys) lying in the plane formed by His-81, His-40, and
Cys-78, is therefore the most reasonable structure for pseudoazurin at
high pH. These results are also consistent with the relationship
between the blue copper active-site transition and the electron
transfer reactivity at an electrode including reduction potential
variations brought about by changes in electron density at the copper
center.
Figure 9: Possible dual electron transfer sites of Achromobacter pseudoazurin.
Pseudoazurin shows a fast electron transfer process at the
4-pyds/Au electrode (k = 3.2
10
cm s
) and slower electron
transfer at the positively charged 4-pydsH
/Au
electrode surface (k
= 6.7
10
cm s
). In the case of
electrochemical experiments on cytochrome c, fast electron
transfer of the protein is believed to be achieved by hydrogen bonding
between the lone-pair electrons on a pyridine moiety at the electrode
and positively charged lysine residues on the protein(37) .
With pseudoazurin, electron transfer is faster at the 4-pyds/Au
electrode than at a pyrolytic graphite (38) or glassy carbon (39) electrode. The slower reaction at the pyrolytic graphite
and glassy carbon electrodes is probably due to the presence of sites
that are less active for electron transfer on a microscopic
scale(40) . At a 4-pydsH
/Au electrode at pH
4.6, the same two determinations were carried out and gave 6.7
10
cm s
and 301 mV versus NHE, respectively. The positively charged electrode surface does
not interact as favorably with the positively charged protein, thus
impeding electron transfer. In recent studies, the electron
self-exchange rate constant for pseudoazurin has been determined by an
NMR line-broadening technique and by cross-reaction studies with Pseudomonas aeruginosa azurin, and a value of 2.8
10
M
s
(average at I = 0.100 M and 25 °C)
was obtained(18) . In the case of azurin (P.
aeruginosa), rapid self-exchange (7.0
10
M
s
at pH 9.0) (41) has been shown to occur via the adjacent hydrophobic patch
through which the His-117 ligand is exposed(26, 42) .
In contrast, pseudoazurin and higher plant plastocyanins have charged
residues at or near the adjacent site and subsequently are less
efficient at self-exchanging due to electrostatic repulsions. It is
interesting to note that the plastocyanin from Anabaena
variabilis, which does not possess an acidic patch, has a much
larger self-exchange rate constant(44) .
In pseudoazurin,
the cause of this repulsion is a number of lysine residues, in
particular Lys-38, which is located in the middle of what might be
described as the adjacent site (at around His-81) and in a similar
position to the M44K residue of the P. aeruginosa azurin
mutant(26, 42) . The latter mutation has a drastic
effect on the rate constant for self-exchange of azurin at low pH
values. Another positive charge on pseudoazurin comes from Lys-77,
which is adjacent to the Cys-78 ligand. This amino acid residue is
located in a similar position to Tyr-83 in plastocyanin, which is in
the middle of the remote acidic patch of the protein. In studies on
cytochrome c from Rhodobacter spheroides,
specific lysine residues have been proposed as a part of the binding
site, their role being to create an optional orientation for electron
transfer(45) , and a similar role for lysine residues in
pseudoazurin is also possible.
It has been reported that the negatively charged A. cycloclastes nitrite reductase complexes strongly with positively charged residues on pseudoazurin(21) . In the case of the blue copper protein amicyanin, a recent electrochemical study has indicated that the reduction potential of the protein is shifted by 73 mV when complex formation with methylamine dehydrogenase occurs(46) . This complex has been confirmed by x-ray structure analysis to have hydrophobic interactions between amicyanin and the enzyme(43) . It is possible that complex formation with nitrite reductase through the positive charges on pseudoazurin induces structural changes at the redox center, so that the reduction potential is tuned for a thermodynamically more favorable electron transfer.