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Stimulation of cytochrome c oxidation by a copper citrate complex
ARCHIVES
OF
BIOCHEMISTRY
AND
Stimulation
BIOPHYSICS
126,
of Cytochrome
c Oxidation
Citrate A. J. DAVISON Department
228-231 (1968)
by a Copper
Complex’ AND R. T. HAMILTON
and Medical Biochemistry, University of Cape Town, and Biochemistry Department, Cornell University, Ithaca, New York Received October 2, 1967; accepted January 23, 1968
of Physiology
A copper-citrate complex acts catalytically to promote the oxidation of cytochrome c by a multistep mechanism The simplest interpretation of the data implies a stepwise reduction of copper-bound oxygen, the reaction showing saturation kinetics with respect to the cat)alyst.
Enzyme-bound copper participates in the cytochrome oxidase catalyzed oxidation of ferrocytochrome c (1). Copper complexes catalyze certain other oxidations (2, 3). A possible catalytic role for copper ions in the oxidation of cytochrome c ((Fe”+) cytochrome c-% (Fe”+) cytochrome c) was therefore investigated. Initial indications of a catalytic effect were found to be influenced by a significant decrease in pH produced by a concentration of copper as low as 6 mM in a 0.1 M phosphate buffer. A citrate-copper complex was therefore tested with the pH previously adjusted to a value of 7.20. The complex was found to produce a marked acceleration of the oxidation of ferrocytochrome c. The dependence of the rate on copper concentration and on cytochrome c concentration was then investigated. EXPERIMENTAL PREPARATION
The oxidation of cytochrome c was initiated by the addition to the cuvette of the copper-citrate solution. The reaction was then followed at 550 rnp using a Beckman Model DB spectrophotometer with a Beckman lo-inch recorder. The cuvette contained 0.4 ml reduced cytochrome c (2.0 mg/ml), graded amounts of copper-citrate solution (0.5 to 0.02 ml), and sufficient Theorell-Stenhagen buffer pH 7.2 to bring the volume to 3 ml. The temperature was controlled at 34.5”. No hydrogen peroxide was detected when a protein-free filtrate of the products of the reaction were tested for ability to oxidize orthotolidine in the presence of peroxidase. Progress of the reaction was kinetically investigated by determining the residual absorbance due to ferrocytochrome c, i.e., the difference, A, between the absorbance at 550 n+ at time t and the absorbance at infinite time. The concentration of ferrocytochrome c then is equal to A/18,500 ~-1 assuming the molar absorptivity determined by Margoliash (5).
OF THE COPPER-CITRATE COMPLEX
RESULTS
The rate of oxidation of cytochrome c was negligible in the absenceof copper, but addition of the copper-citrate solution produced an immediate and rapid oxidation (Fig. 1). Progress of the oxidation was analyzed by the differential plot method (log (CM/&) us log A) (6) and straight-line graphs were obtained (Fig. ZA). The slopes were determined by least-squares analysis, which showed that the reaction is second order with respect to ferrocytochrome c
A solution with a final concentration of 290 mM with respect to copper and a pH of 7.2 was prepared by neutralizing copper sulfate in the presence of 145 rnM citric acid, which by complex formation prevented precipitation of the copper. Cytochrome c (Grade 1, Seravac Laboratories, Maidenhead, England) of 95’% purity was reduced using H,/Pd (4). 1 Supported by grants from the South African Council for Scientific and Industrial Research, and the William Adam Jolly Fund. 228
STIMULATION
OF
CYTOCHROME
229
c OXIDATION TABLE
I
EFFECT OF COPPER CONCENTRATION ON RATE AND ORDER OF RE.~cTION Final copper concentration as total Cu(I1) (mar)
0
1000 Time
38.7 29.0 19.3 9.8 5.9 1.9
2000 (sec.)
Order with respect to cytochrome c
1.99 2.03 2.19 2.32 2.37 3.15
f f f f f f
Apparent second-order rate constant Initial velocity liters moles-’ pmoles, ‘-I, set-’ set-1 ’
.07 .06 .08 .08 .07 .lO
37.2 34.8 31.5 27.3 23.4 19.7
9.5 8.9 8.3 7.2 6.0 5.0
x x x x x x
104 104 104 lo4 lo4 104
FIG. 1. Effect on the rate of oxidation of ferrocytochrome c of the addition (arrow at 690 set) of the copper-citrate complex (final concentration 29.0 mM with respect to copper).
-3.
-3.
-4. -0.6
-0.4 -0.2 log Absorbance
0
(Cu >mM FIG. 3. The rate (mmoles. copper (Cu).
6
dependence l-l.sec-1)
of the initial reaction on the concentration of
(c2+), that is --- d( c”) at
IO
1000 Time
2000 (se@
FIG. 2. A, Differential plot analysis of the dependence of reaction rate on ferrocytochrome c concentration. Data from Fig. 1. Slope equals 2.03 f 0.06. B, Plot showing second-order ratedependence on ferrocytochrome c concentration, from the time of addition of the copper-citrate complex. Data from Fig. 1.
= lc(cZf)”
(1)
This was confirmed by plotting l/A us t (Fig. 2B). The rates and orders are listed in Table I. The slight increase in the order at the lower copper-citrate concentrations is probably not due to experimental error and deserves further invest,igation. DISCUSSION
The kinetics of the reaction are unexpectedly complex since although a cupric ion can accept only a single electron from ferrocytochrome c, the second-order depend-
DAVISON
230
AND
I.0
0.5 x IO3
HAMILTON
catalytic role for copper. Figure 4 shows that the experimental data closely follow the relationship (Cu)/k = &(cu) + k3. Substituting for Ic in equation I and introducing the arbitrary constant kl = 1, we obtain, as the over-all rate equat)ion for the reaction:
-- d( c’+) = kl(C2+)2(CU) dt
FIG. 4. The dependence of the apparent secondorder rate constant (k) on copper concentration, plotted in a manner analogous to the S/v us S plot for enzyme kinetic data. The units for (Cu)/k are moles2~1-2~sec.
ence with respect to cytochrome c implies two reactions with ferrocytochrome c prior to the rate-determining step. Presumably after accepting a single electron from ferrocytochrome c, it is necessary for the copper to transfer the electron to a suitable acceptor before it can react with another molecule of ferrocytochrome c. Evidently the copper is acting as a catalyst of the auto-oxidation of cytochrome c, and not merely as an oxidizing agent. The second reaction must be with a copper-acceptor complex, since otherwise it would be merely a repetition of the first reaction and the kinetics would be first order. This mechanism is similar to that suggested by Frieden (2) for the copper-catalyzed oxidation of ascorbate except that whereas Frieden proposed reduction of the copper-oxyion by a cuprous ion, the secondorder dependence on cytochrome c suggests that in this case reduction of the copperoxyion occurs by a second ferrocytochrome c molecule rather than by a cuprous ion. Similar stepwise mechanisms have been proposed for the enzymatic oxidation of cytochrome c by cytochrome oxidase (7, 8, 9). The dependence of the initial reaction rate (k) on copper concentration is shown in Fig. 3 and constitutes further evidence of a
/c,(Cu>
+
k3
(2)
This equation implies saturation kinetics with respect to catalyst, a behavior inverse to normal Michaelis-Menton behavior which may be expected to occur when catalyst concentration exceeds that of substrate, as in this instance. Values i&/k2 (i.e., maximum velocity/(c2+)2) and k&z (“Michaelis constant”) are respectively 40 moles-l .l.sec-I, and 4 X W3 M. The latter value is predictably higher (about 1,000 times) than the Michaelis constant for the cytochrome c-cytochrome oxidase reaction. It has yet to be established whether or not the participation of cytochrome c in the cytochrome oxidase reaction is multistep, as it appears to be in this model system. The cyt’ochrome oxidase reaction is normally first order with respect,to ferrocytochrome c, indicating that if subsequent reactions of the enzyme with cytochrome c occur, they occur after the rate-determining step. Wainio et al. have, however, recently shown that, at an early stage the cytochrome oxidase reaction is second order (10). They interpret this as possible evidence for participation of the enzyme in the reaction as a dimer. The behavior of the system presented here suggests that the possibility of a sequential reaction with cytochrome c in their system should be considered also, as should be the possibility of presteady state conditions during which the oxidase concentration might be decreasing. REFERENCES 1. GRIFFITHS,
D. E., AND WHARTON, D. Biol. Chem. 236, 1857 (1961). 2. FRIEDEN, E., OSAKI, S., AND KOBAYASNI, J. Gen. Physiol. 49, 213 (1966). 3. PECHT, I., LEVITASKI, A., AND ANBAS, J. Am. Chem. Sac. 89, 1587 (1967).
C., J. H,, W,,
STIMULATION 4. SMITH,
L.,
Arch.
Biochem.
OF Biophus.
CYTOCHROME 60,
285
(1954). 5. MARGOLI.4SH, E., Biochem. J. 66, 535 (1954). 6. LAIDLER, E., “Chemical Kinetics,” 2nd Edit., McGraw Hill, New York (1965). 7. Lu VALLE, J. E., AND GODDARD, R. J., Quart. Rev. Biol. 23, 197 (1948).
c OXIDATION
231
8. DAVISON, A. J., AND WAINIO, W. W., Federation Proc. 23, 1322 (1964). 9. FRIDOVICH, I., J. Biol. Chem. 236, 1836 (1961). 10. WAINIO, W. W., GREBNER, D., AND O’FARRELL, H., Symp. on Structural and Chemical Aspects of Cytochromes, Univ. of Tokyo Press 0.38 (1967).
OF
BIOCHEMISTRY
AND
Stimulation
BIOPHYSICS
126,
of Cytochrome
c Oxidation
Citrate A. J. DAVISON Department
228-231 (1968)
by a Copper
Complex’ AND R. T. HAMILTON
and Medical Biochemistry, University of Cape Town, and Biochemistry Department, Cornell University, Ithaca, New York Received October 2, 1967; accepted January 23, 1968
of Physiology
A copper-citrate complex acts catalytically to promote the oxidation of cytochrome c by a multistep mechanism The simplest interpretation of the data implies a stepwise reduction of copper-bound oxygen, the reaction showing saturation kinetics with respect to the cat)alyst.
Enzyme-bound copper participates in the cytochrome oxidase catalyzed oxidation of ferrocytochrome c (1). Copper complexes catalyze certain other oxidations (2, 3). A possible catalytic role for copper ions in the oxidation of cytochrome c ((Fe”+) cytochrome c-% (Fe”+) cytochrome c) was therefore investigated. Initial indications of a catalytic effect were found to be influenced by a significant decrease in pH produced by a concentration of copper as low as 6 mM in a 0.1 M phosphate buffer. A citrate-copper complex was therefore tested with the pH previously adjusted to a value of 7.20. The complex was found to produce a marked acceleration of the oxidation of ferrocytochrome c. The dependence of the rate on copper concentration and on cytochrome c concentration was then investigated. EXPERIMENTAL PREPARATION
The oxidation of cytochrome c was initiated by the addition to the cuvette of the copper-citrate solution. The reaction was then followed at 550 rnp using a Beckman Model DB spectrophotometer with a Beckman lo-inch recorder. The cuvette contained 0.4 ml reduced cytochrome c (2.0 mg/ml), graded amounts of copper-citrate solution (0.5 to 0.02 ml), and sufficient Theorell-Stenhagen buffer pH 7.2 to bring the volume to 3 ml. The temperature was controlled at 34.5”. No hydrogen peroxide was detected when a protein-free filtrate of the products of the reaction were tested for ability to oxidize orthotolidine in the presence of peroxidase. Progress of the reaction was kinetically investigated by determining the residual absorbance due to ferrocytochrome c, i.e., the difference, A, between the absorbance at 550 n+ at time t and the absorbance at infinite time. The concentration of ferrocytochrome c then is equal to A/18,500 ~-1 assuming the molar absorptivity determined by Margoliash (5).
OF THE COPPER-CITRATE COMPLEX
RESULTS
The rate of oxidation of cytochrome c was negligible in the absenceof copper, but addition of the copper-citrate solution produced an immediate and rapid oxidation (Fig. 1). Progress of the oxidation was analyzed by the differential plot method (log (CM/&) us log A) (6) and straight-line graphs were obtained (Fig. ZA). The slopes were determined by least-squares analysis, which showed that the reaction is second order with respect to ferrocytochrome c
A solution with a final concentration of 290 mM with respect to copper and a pH of 7.2 was prepared by neutralizing copper sulfate in the presence of 145 rnM citric acid, which by complex formation prevented precipitation of the copper. Cytochrome c (Grade 1, Seravac Laboratories, Maidenhead, England) of 95’% purity was reduced using H,/Pd (4). 1 Supported by grants from the South African Council for Scientific and Industrial Research, and the William Adam Jolly Fund. 228
STIMULATION
OF
CYTOCHROME
229
c OXIDATION TABLE
I
EFFECT OF COPPER CONCENTRATION ON RATE AND ORDER OF RE.~cTION Final copper concentration as total Cu(I1) (mar)
0
1000 Time
38.7 29.0 19.3 9.8 5.9 1.9
2000 (sec.)
Order with respect to cytochrome c
1.99 2.03 2.19 2.32 2.37 3.15
f f f f f f
Apparent second-order rate constant Initial velocity liters moles-’ pmoles, ‘-I, set-’ set-1 ’
.07 .06 .08 .08 .07 .lO
37.2 34.8 31.5 27.3 23.4 19.7
9.5 8.9 8.3 7.2 6.0 5.0
x x x x x x
104 104 104 lo4 lo4 104
FIG. 1. Effect on the rate of oxidation of ferrocytochrome c of the addition (arrow at 690 set) of the copper-citrate complex (final concentration 29.0 mM with respect to copper).
-3.
-3.
-4. -0.6
-0.4 -0.2 log Absorbance
0
(Cu >mM FIG. 3. The rate (mmoles. copper (Cu).
6
dependence l-l.sec-1)
of the initial reaction on the concentration of
(c2+), that is --- d( c”) at
IO
1000 Time
2000 (se@
FIG. 2. A, Differential plot analysis of the dependence of reaction rate on ferrocytochrome c concentration. Data from Fig. 1. Slope equals 2.03 f 0.06. B, Plot showing second-order ratedependence on ferrocytochrome c concentration, from the time of addition of the copper-citrate complex. Data from Fig. 1.
= lc(cZf)”
(1)
This was confirmed by plotting l/A us t (Fig. 2B). The rates and orders are listed in Table I. The slight increase in the order at the lower copper-citrate concentrations is probably not due to experimental error and deserves further invest,igation. DISCUSSION
The kinetics of the reaction are unexpectedly complex since although a cupric ion can accept only a single electron from ferrocytochrome c, the second-order depend-
DAVISON
230
AND
I.0
0.5 x IO3
HAMILTON
catalytic role for copper. Figure 4 shows that the experimental data closely follow the relationship (Cu)/k = &(cu) + k3. Substituting for Ic in equation I and introducing the arbitrary constant kl = 1, we obtain, as the over-all rate equat)ion for the reaction:
-- d( c’+) = kl(C2+)2(CU) dt
FIG. 4. The dependence of the apparent secondorder rate constant (k) on copper concentration, plotted in a manner analogous to the S/v us S plot for enzyme kinetic data. The units for (Cu)/k are moles2~1-2~sec.
ence with respect to cytochrome c implies two reactions with ferrocytochrome c prior to the rate-determining step. Presumably after accepting a single electron from ferrocytochrome c, it is necessary for the copper to transfer the electron to a suitable acceptor before it can react with another molecule of ferrocytochrome c. Evidently the copper is acting as a catalyst of the auto-oxidation of cytochrome c, and not merely as an oxidizing agent. The second reaction must be with a copper-acceptor complex, since otherwise it would be merely a repetition of the first reaction and the kinetics would be first order. This mechanism is similar to that suggested by Frieden (2) for the copper-catalyzed oxidation of ascorbate except that whereas Frieden proposed reduction of the copper-oxyion by a cuprous ion, the secondorder dependence on cytochrome c suggests that in this case reduction of the copperoxyion occurs by a second ferrocytochrome c molecule rather than by a cuprous ion. Similar stepwise mechanisms have been proposed for the enzymatic oxidation of cytochrome c by cytochrome oxidase (7, 8, 9). The dependence of the initial reaction rate (k) on copper concentration is shown in Fig. 3 and constitutes further evidence of a
/c,(Cu>
+
k3
(2)
This equation implies saturation kinetics with respect to catalyst, a behavior inverse to normal Michaelis-Menton behavior which may be expected to occur when catalyst concentration exceeds that of substrate, as in this instance. Values i&/k2 (i.e., maximum velocity/(c2+)2) and k&z (“Michaelis constant”) are respectively 40 moles-l .l.sec-I, and 4 X W3 M. The latter value is predictably higher (about 1,000 times) than the Michaelis constant for the cytochrome c-cytochrome oxidase reaction. It has yet to be established whether or not the participation of cytochrome c in the cytochrome oxidase reaction is multistep, as it appears to be in this model system. The cyt’ochrome oxidase reaction is normally first order with respect,to ferrocytochrome c, indicating that if subsequent reactions of the enzyme with cytochrome c occur, they occur after the rate-determining step. Wainio et al. have, however, recently shown that, at an early stage the cytochrome oxidase reaction is second order (10). They interpret this as possible evidence for participation of the enzyme in the reaction as a dimer. The behavior of the system presented here suggests that the possibility of a sequential reaction with cytochrome c in their system should be considered also, as should be the possibility of presteady state conditions during which the oxidase concentration might be decreasing. REFERENCES 1. GRIFFITHS,
D. E., AND WHARTON, D. Biol. Chem. 236, 1857 (1961). 2. FRIEDEN, E., OSAKI, S., AND KOBAYASNI, J. Gen. Physiol. 49, 213 (1966). 3. PECHT, I., LEVITASKI, A., AND ANBAS, J. Am. Chem. Sac. 89, 1587 (1967).
C., J. H,, W,,
STIMULATION 4. SMITH,
L.,
Arch.
Biochem.
OF Biophus.
CYTOCHROME 60,
285
(1954). 5. MARGOLI.4SH, E., Biochem. J. 66, 535 (1954). 6. LAIDLER, E., “Chemical Kinetics,” 2nd Edit., McGraw Hill, New York (1965). 7. Lu VALLE, J. E., AND GODDARD, R. J., Quart. Rev. Biol. 23, 197 (1948).
c OXIDATION
231
8. DAVISON, A. J., AND WAINIO, W. W., Federation Proc. 23, 1322 (1964). 9. FRIDOVICH, I., J. Biol. Chem. 236, 1836 (1961). 10. WAINIO, W. W., GREBNER, D., AND O’FARRELL, H., Symp. on Structural and Chemical Aspects of Cytochromes, Univ. of Tokyo Press 0.38 (1967).