The reduction of Cu(330) in Rhus vernicifera laccase by chromous ion is 30% faster than reduction of Cu(614) at room temperature [pH 4.8, µ = 0.1 (NaCl)], and two parallel first-order paths, attributed to heterogeneity of the protein, are observed at both wavelengths. The reactions of stellacyanin, spinach and French-bean plastocyanins, and cytochrome c with chromous ion under similar conditions are faster than that with laccase by factors of 102 to 104, and are first order in protein concentration. Comparison of rates and activation parameters for the reduction of "blue" copper in laccase, stellacyanin, and the two plastocyanins indicates that reduction of the Cu(614) site in laccase may occur by intramolecular electron transfer from one of the Cu(330) sites. Our value of  ΔH (17.4 kcal/mol) for the chromous ion reduction of cytochrome c is consistent with a mechanism in which major conformational changes in the protein must accompany electron transfer

Dawson, J. W.

Gray, H. B.

Holwerda, R. A.

Westhead, E. W.

English

Caltech Authors - Main

Proc. Nat. Acad. Sci. USA
Vol. 69, No. 1, pp. 30-33, January 1972
Kinetics of the Reduction of Metalloproteins by Chromous Ion
(laccase/cytochrome c/plastocyanins/temperature/rate constants)
J. W. DAWSON, H. B. GRAY*, R. A. HOLWERDA, AND E. W. WESTHEADt
The Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, Calif. 91109
Contributed by H. B. Gray, October 27, 1971
ABSTRACT The reduction of Cu(330) in Rhus verni-
cifera laccase by chromous ion is 30% faster than reduction
of Cu(614) at room temperature [pH 4.8, u = 0.1 (NaCl)],
and two parallel first-order paths, attributed to hetero-
geneity of the protein, are observed at both wavelengths.
The reactions of stellacyanin, spinach and French-bean
plastocyanins, and cytochrome c with chromous ion under
similar conditions are faster than that with laccase by
factors of 102 to 104, and are first order in protein concentra-
tion. Comparison of rates and activation parameters for
the reduction of "blue" copper in laccase, stellacyanin,
and the two plastocyanins indicates that reduction of the
Cu(614) site in laccase may occur by intramolecular elec-
tron transfer from one of the Cu(330) sites. Our value of
AlH (17.4 kcal/mol) for the chromous ion reduction of
cytochrome c is consistent with a mechanism in which
major conformational changes in the protein must ac-
company electron transfer.
We have begun an extensive investigation of the mechanisms
of the reduction/oxidation (redox) reactions between metal-
loproteins and inorganic inner- and outer-sphere reducing
agents. Our initial research has been concerned with the re-
duction rates of horse heart cytochrome c and the copper
proteins Rhus vernicifera laccase, stellacyanin, and plasto-
cyanins from spinach and French-bean. Cytochrome c is
particularly valuable as a model protein oxidant because the
molecular structures of both oxidized and reduced forms have
recently been determined (1, 2). Although as yet no direct
x-ray structural data are available for the copper proteins,
extensive spectroscopic studies have characterized the dif-
ferent types of Cu2+ sites (3).
This paper compares the kinetic behavior of cytochrome c
and the copper proteins with aqueous chromium(II). These
reactions should yield valuable mechanistic information be-
cause there is substantial literature on Cr(II) reductions of
model transition metal complexes (4). Furthermore, Kowal-
sky has shown that some of the Cr(III) product remains
bound to the protein after the chromous reduction of cyto-
chrome c (5). Thus, the substitution-inert Cr(III) may label
one or more key reduction sites in reactions with cytochrome
c and possibly the copper proteins.
MATERIALS AND METHODS
Distilled deionized water was used for kinetics experiments.
Concentrated Cr(II) solutions were made up from high-
purity Cr metal (Alfa Inorganics) and a slight excess of deoxy-
genated hydrochloric acid. Deoxygenation was accomplished
by purging solutions with nitrogen, which had been passed
through towers containing chromous solutions to remove
oxidizing impurities. Dilute chromous solutions were pre-
pared by standard syringe techniques and were continuously
purged with nitrogen. These solutions were assayed by re-
acting aliquots with excess standard KMnO4 in 0.2 M H2SO4
and determining residual MnO4- spectrophotometrically at
545 nm (e..s = 2340 M-1 cm-'). Analyses for total chromium
by the basic peroxide procedure (6) agreed well with results
of Cr(II) determinations.
Reagent grade sodium chloride was used to supplement the
ionic strength of chromous and protein solutions to 0.1.
Plastocyanins from spinach and French-bean (Phaseolus
vulgaris, var. Prince) were isolated and purified by modifica-
tions of the method used by Katoh et al., (7) for spinach
plastocyanin. The chloroplasts were obtained with a Waring
blendor rather than by grinding in a mortar, and the purifica-
tion procedure as improved by gel filtration (Bio-Gel P-60 or
P-30) of protein solutions. Samples used for kinetics experi-
ments had A278/A597 ratios of 1.24 (spinach) and 1.48 (bean).
Stellacyanin and laccase were extracted from the latex of the
Japanese lacquer tree Rhus vernicifera using the method of
Reinhammar (8); the A2ws/Aw4 ratio (stellacyanin) was 5.6 and
the A280/A614 ratio (laccase) was 15.3. The horse-heart cyto-
chrome c was from the same sample used for the x-ray struc-
tural determination (1), and was kindly donated by Dr. T.
Takano.
After removal from liquid-nitrogen storage, the copper
protein solutions used in this work were dialyzed for 6 hr
against two changes of deionized water; they were diluted to
yield an absorbance change at about 600 nm of 0.15-0.30
(2-cm path) on reduction.
The pH of protein solutions was adjusted with dilute HCl
and a Corning model 12 pH meter before degassing. The pH
of dilute chromous solutions was monitored continuously
with the pH meter and a Thomas combination electrode,
which was inserted through a rubber serum stopper in one
neck of the storage flask. Deoxygenated dilute NaOH solu-
tion, injected with a syringe, was used to bring Cr(II) solu-
tions to the desired pH.
Chromous reductions of "blue" copper proteins and of
cytochrome c were followed spectrophotometrically with a
Durrum-Gibson stopped flow apparatus. The copper absorp-
tion maxima at 597, 604, and 614 nm were used to study the
reduction of the two plastocyanins, stellacyanin, and laccase,
respectively. Data for Rhus laccase were also collected at 330
30
* To whom correspondence should be addressed.
t Present address: Department of Biochemistry, University of
Massachusetts, Amherst, Mass. 01002
Proc. Nat. Acad. Sci. USA 69 (1972)
nm. The production of ferrocytochrome c was observed at
550 nm.
Chromous and protein solutions were transferred from
storage flasks directly into the air-tight, stopped-flow syringes
with Kel-F and Teflon fittings (Hamilton Co., Whittier,
Calif.) and stainless-steel hypodermic needles. The stopped-
flow syringes were immersed in a small bath containing water
circulated from a constant temperature reservoir (Forma
Scientific). Temperature control was estimated to be ±t0.20C.
Pseudo first-order conditions for the metalloproteins were
used, a 50- to 100-fold excess of reducing agent being present
in most experiments ([Cr(II) ]o _ 5 X 10-1 M). Stopped-flow
traces were analyzed through the usual plots of log (A, - Ax.)
against time. Rate constants were evaluated assuming a first-
order rate dependence on chromous ion; this assumption will
be tested in experiments presently underway.
RESULTS
As shown in Fig. 1, the reduction of cytochrome c by an ex-
cess of Cr(II) follows a pseudo first-order rate dependence
through more than 90% of the reaction. This plot is typical
of the data obtained at all temperatures and for all of the
metalloproteins studied here, except Rhus laccase.
By contrast, the reduction of the "blue" copper of laccase
(614 nm) does not follow simple first-order kinetics (Fig. 2).
Plots of log (A, - A.) against time were analyzed by ex-
trapolation of the linear portion of the curve, from the latter
part of the reaction, back to the ordinate. The difference be-
tween this extrapolated line and the observed curve was
plotted on a semilog scale. The consistent linearity of this
secondary plot is in accord with a mechanism in which the loss
of absorbance at 614 nm is the result of two parallel first-
order processes. Laccase data at 330 nm also show two rate
0.0
-0.5
- 1.0
0
-1.5-
-2.0
I
0 8 1 6 24
mseconds
FIG. 1. Typical plot of log (A., - A) against time for
reduction of cytochrome c by chromous ion. T = 37.80C, (pH
4.2), [Cr(II)]o = 5.4 X 10-3M.
<
-1.51-.O . o
-2.0
0 2 3 4 5 6
Seconds
FIG. 2. Plot of log (At - A.) against time for the reduction
of Rhus vernicifera laccase by chromous ion. T = 40.20C, (pH
4.8), [Cr(II)]o = 5.4 X 10-3 M. Reaction followed at 614 nm.
Triangles mark the derivative plot of log (At -A0) for the fast
initial phase of the reaction.
constants above 230C, but at lower temperatures there was
no apparent deviation from the simple first-order line.
By comparing the oridnate intercepts of the lines cor-
responding to fast and slow rate constants with the total
change in absorbance from fully oxidized to fully reduced
protein, it was possible to estimate that throughout the
temperature range the fast-reducing component was respon-
sible for the loss of about two-thirds of the total blue color at
614 nm.
The rate data for the chromous reduction of cytochrome c
at pH 4.2 are shown in Fig. 3a as an Eyring plot of log (kiT)
against 1/T. Thermodynamic activation parameters cal-
culated for this and all other reactions are summarized in
Table 1, along with rate constants at room temperature
estimated from Eyring plots.
Eyring plots for the chromous ion reduction of laccase at
pH 4.8 are shown in Fig. 3b. The data for the slow reactions
at 614 and 330 nm are so similar that, to within the limits of
experimental error, they might be attributed to the same re-
action. The fast reaction data at 614 nm at the low tempera-
ture extreme consistently deviate from the straight line plot
of log (kIT) against 1/T plot that best fits all the other points.
Further study of this anomaly is necessary.
Spinach and bean plastocyanins showed rate constants of
the order of 104 M-1 sec', with little variation with tempera-
ture. The bean plastocyanin data show considerable scatter,
but no consistent trend with temperature. Consecutive traces
obtained at 31.800C and 4.00C show less than a 5% difference
in rate. We estimate AHR to be less than 1 kcal/mol.
Because stellacyanin reacts so rapidly with Cr(II), it was
necessary to work with low concentrations of chromous ion
Reduction of Metalloproteins 31
32 Biochemistry: Dawson et al.
TABLE 1. Rate data for the chromium(II) reduction
of metalloproteins*
-~~~ ~ ~ ~ ~~~~~0LtS?
k (250C) AHt (entropy
Protein M-1 sec' (kcal/mol) units) pH
Cytochrome c 1.2 X 104 17.4 +18 4.2
Rhus laccase
614 nm-
Fast 24 12.9 -9 4.8
Slow 7 13.8 -9 4.8
330 nm-
Fast 32 13.4 -7 4.8
Slow 5 15.2 -4 4.8
Spinach
plastocyanin 3.3 X 104 1.3 -34 4.2
Bean
plastocyanin 1 X 104 1 4.2
Stellacyanin 6 X 105 - - 4.2
*
, = 0.1 (NaCI); estimated uncertainties are 41 kcal/mol
in AHT and 42 entropy units in ASt.
and to use the shortest time scale of the instrument. A second-
order rate constant of 6 X 105 M-1 sec-' (4C, pH 4.2) was
estimated, and little increase was noted at 200C.
DISCUSSION
Study of the temperature dependence of cytochrome c re-
ductions by small molecules has shown that the hydroquinone
reduction is sensitive to pH and ionic strength, with Ea =
20 kcal/mol, ASt +15 entropy units [k = 3.7 M-l sec,
12'C (pH 7.0); ,u = 0.17] (9). Reduction of cytochrome c
by the semiquinone radical is about 105-times faster. A
nonlinear Arrhenius plot has been found for the reduction of
cytochrome c by ascorbate (10); the slope of the lower tem-
perature region corresponds to E, = 25-30 kcal/mol
(pH 7.0, 0.1 M phosphate buffer). By contrast, the rate of
reduction of the "low-pH form" of cytochrome c by ascorbate
was found to be almost temperature independent. A slower
reacting form appeared to be converted to an active form,
at a rate characterized by Ea = + 19 kcal/mol (11). Very
fast reductions of cytochrome c by tetrachlorohydroquinone
(k = 6 X 106 M-1 sec1) (9) and by hydrated electrons (12)
have been reported. The ferrocyanide reduction of cytochrome
c proceeds at a rate very similar to that observed for the
chromous reduction (13).
The pulsed radiolysis work of Land and Swallow (12)
showed that transfer of an electron to the iron atom of cyto-
chrome c is accompanied by the full change in absorbance at
550 nm. The final change in absorbance at shorter wave-
lengths is completed much more slowly, indicating that after
the initial reduction of iron the protein accommodates itself
to the new oxidation state in a reaction significantly slower
than electron transfer. This secondary reaction was found to
have an activation energy of 20 kcal/mol, which is the same
as that found for the reduction of cytochrome c by hydro-
quinone and similar to our value of ARH (17.4 kcal/mol) for
the reduction by chromous ion.
It seems possible that with less reactive species than hy-
drated electrons, electron transfer cannot take place without
simultaneous conformational changes in the protein. Such
a mechanism would explain the similarity of activation en-
ergy terms for various reducing agents. Less efficient reducing
agents, of course, might have additional barriers to reaction,
but results with other facile one-electron transition metal
reductants should clarify whether conformational changes in
the protein are rate-determining in the electron transfer
reactions of cytochrome c.
2.0h
H-
0
3.0
0.0 r-
3.63.2 3.4
1/T (OK-I) x 103
-1.0 [-
-
0
-2.0
-3.0 I
3.0 3.63.2 3.4
1/T (0KI) x 103
FIG. 3. (a) Eyring plot of rate data for the chromous ion re-
duction of cytochrome c at pH 4.2, I' = 0.1.
(b) Eyring plots of rate data for the chromous ion reduc-
tion of Rhus vernicifera laccase at pH 4.8,,1 = 0.1. A- - -, fast
reaction, 330 nm; 0---, fast reaction, 614 nm; 0- - -, slow reac-
tion, 330 nm; 0- - -, slow reaction, 614 nm.
a
0
1.0
b
0
Proc. Nat. Acad. Sci. USA 69 (1972)
Reduction of Metalloproteins 33
The unusually favorable AS~value for the chromous ion re-
duction of cytochrome c is similar to that calculated for the
reaction with hydroquinone (9). Such a large positive AS* is
remarkable in view of the negative contribution expected (14)
from the solvation changes associated with formation of a
transition state involving the strongly basic ferricytochrome c
with a dipositive ion at pH 4.2. One possible explanation is
that the protein conformational change required to reach the
transition state places the hydrophobic side chain of phenyl-
alanine 82 substantially into the heme crevice, where it is
found in the reduced protein (2), thereby breaking up a sig-
nificant amount of ordered water structure.
Most studies (3) of laccase have used the protein derived
from Polyporus versicolor, rather than from Rhus vernicifera.
The Polyporus enzyme has four copper atoms; one is respon-
sible forthe 614-nm absorption ("Type 1"), one is detected only
from its electron paramagnetic resonance (EPR) spectrum
("Type 2"), and two others ("Type 3" copper), unobservable
in EPR spectra, are responsible for the absorption shoulder at
330 nm. There is evidence for cooperative behavior among
the copper atoms of this protein; it appears that ion binding
at the Type-2 copper inhibits enzymatic activity. Quinol
reduces Type-1 copper most rapidly, whereas azide reduces
the Type-2 copper first (15). Redox titrations of the molecule
show that in the absence of an inhibitor, Type-1 and Type-3
copper atoms are reduced simultaneously.
The Rhus vernicifera laccase has only recently been given
considerable attention. In general it seems very similar to
the fungal enzyme (16). Titration of oxidized laccase by
ascorbate, or the reverse titration with hydrogen peroxide,
showed that the "blue" copper has an E' value of 0.045-V
lower than that of Cu(330) at 250C (pH 7.0) (17). The two
types of copper appear to be in redox equilibrium, but it is
not certain that Cu(614) and Cu(330) iDteract intramolecu-
larly. Our finding that Cr(JI) reduces Cu(330) about 30%
faster than Cu(614) is in accord with the above-mentioned
data. The simplest explanation for the biphasic curves found
for the reduction of laccase, followed either at 614 or 330 nm,
is heterogeneity of the laccase preparation. Fungal laccase
preparations have been shown to be heterogeneous from the
EPR spectra of Type-2 copper (18). The protein has not
been resolved into separate entities. The similarity of our re-
sults at 614 and 330 nm for Rhus laccase implies that the effect
of the heterogeneity is felt by both types of copper. Why
the heterogeneity is not observed at 330 nm at lower tem-
peratures is not readily understandable.
The rate of chromous ion reduction of the "blue" copper in
laccase is slower than reduction rates observed for the other
copper proteins by factors of 102-104. Comparison of activa-
tion parameters for the laccase and spinach plastocyanin
reductions reveals a dramatic difference in both AHt and
AS . A possible explanation for this behavior is that reduction
of Cu(614) by Cr(JI) takes place intramolecularly only after
some Cu(330) has been reduced. Thorough investigation of
the kinetic order with respect to Cr(II) should be helpful in
discussing this possibility, as a mechanism involving intra-
(or inter-) molecular electron transfer steps may not yield a
simple experimental (Cr2+) dependence. Experiments with
ions inhibiting reduction of Cu(330) potentially could show
if inhibition of Cu(330) affects the reduction of Cu(614) and
provide an estimate of the rate of direct reaction, if any,
between chromium (II) and Cu(614) in laccase.
We thank J. M. Schredder and B. Dohner for technical as-
sistance and Dr. R. E. Dickerson for useful discussions. Helpful
comments on our manuscript were provided by Dr. L. Bennett.
This work was supported by the National Science Foundation
(H.B.G.), and in part by the USPHS grants GM 14945 and AM
11157 (E.W.W.). R.A.H. thanks the N.S.F. for a Graduate
Fellowship (1970-71). This is contribution No. 4367 from the
Arthur Amos Noyes Laboratory.
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Samson, L., Cooper, A. & Margoliash, E. (1971) J. Biol.
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2. Dickerson, R. E., Takano, T. & Kallai, 0. B., Proc. Cold
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Proc. Nat. Acad. Sci. USA 69 (1972)


Kinetics of the reduction of metalloproteins by chromous ion

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Kinetics of the reduction of metalloproteins by chromous ion

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