©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Design of a Ruthenium-Cytochrome c Derivative to Measure Electron Transfer to the Initial Acceptor in Cytochrome c Oxidase (*)

(Received for publication, September 6, 1994; and in revised form, November 18, 1994)

Lois M. Geren (1) James R. Beasley (2)(§) Bryan R. Fine (2) Aleister J. Saunders (2)(¶) Sharon Hibdon (1) Gary J. Pielak (2) Bill Durham (1) Francis Millett (1)(**)

From the  (1)Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701 and (2)the Departments of Chemistry and Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-3290

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4`methylbipyridine) (bisbipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 ± 2.0) times 10^5 s, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 ± 0.2) times 10^6 s. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH 7) resulted in electron transfer from photoreduced heme c to Cu(A) with a rate constant of (6 ± 2) times 10^4 s, followed by electron transfer from Cu(A) to heme a with a rate constant of (1.8 ± 0.3) times 10^4 s. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 ± 0.4) times 10^7M s at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.


INTRODUCTION

Cytochrome c oxidase is a redox-linked proton pump that transfers electrons from cytochrome c to molecular oxygen(1, 2, 3) . Three of the redox active metal centers, heme a, heme a(3), and Cu(B), are located in subunit I, and their ligands have been identified by extensive mutagenesis experiments on Rhodobacter sphaeroides cytochrome c oxidase (4, 5) . Cu(A) is located in subunit II and is liganded by Cys, Cys, His, and His(6, 7, 8, 9) . The Cu(A) center has been suggested to consist of a mixed-valence, binuclear Cu(1.5), Cu(1.5) cluster rather than a single copper atom(10, 11, 12) . The complex between cytochrome c and cytochrome c oxidase is stabilized by electrostatic interactions between the highly conserved lysines surrounding the heme crevice of cytochrome c and four or more carboxylates on subunit II(13, 14, 15, 16, 17) . One of these carboxylates, Glu-198, is located between the Cu(A) ligands Cys and Cys. The close spatial relation between Cu(A) and the binding site led to the suggestion that Cu(A) might accept electrons directly from cytochrome c(17) .

The characterization of the electron transfer reaction from cytochrome c to the initial acceptor in cytochrome c oxidase has been a difficult problem. Stopped-flow spectroscopy has been used to study the bimolecular kinetics(18, 19) , but does not appear to have sufficient time resolution to measure the initial electron transfer step within the complex. Flow-flash photolysis studies of the complex between ferrocytochrome c and reduced-CO-inhibited cytochrome c oxidase indicated that heme c transferred an electron to Cu(A) with a rate constant greater than 7 times 10^4 s(20) . In contrast, a rate constant of only 630 s was observed for electron transfer from cytochrome c to heme a in cytochrome c oxidase using a photoreduced flavin to initiate the reaction(21) . Pan et al.(22) used our new ruthenium photoexcitation technique(23, 24, 25, 26, 27, 28, 29, 30) to study electron transfer within complexes between cytochrome c oxidase and derivatives labeled at specific lysines on the backside of horse cytochrome c with Ru(bipyridine)(3). The Ru(II) group is photoexcited to a metal-to-ligand charge-transfer state, Ru(II*), which is a strong reducing agent and rapidly transfers an electron to heme c. The photoreduced heme c was found to initially transfer an electron to Cu(A) with a rate constant greater than approximately 1 times 10^5 s, followed by electron transfer from Cu(A) to heme a with a rate constant of 2 times 10^4 s. It was not possible to measure the rate constant for electron transfer from heme c to Cu(A) for these derivatives, probably because the rate for electron transfer from Ru(II*) to heme c is not fast enough compared to the subsequent electron transfer to Cu(A).

In this study we have prepared a new Ru-cyt c derivative specifically designed to measure the initial electron transfer reaction from cytochrome c to cytochrome c oxidase. Two design criteria had to be satisfied to achieve this goal. First, the Ru-cyt c derivative must interact with cytochrome c oxidase in the same fashion as wild-type cytochrome c. Second, the rate of electron transfer from Ru(II*) to the heme group must be fast compared to the rate of electron transfer from the heme group to the initial acceptor in cytochrome c oxidase. This requires an efficient pathway for electron transfer from Ru(II*) to the heme group, and optimum redox properties for Ru(II*). Beratan et al.(31) have identified an efficient pathway for electron transfer from residue 39 on the backside of cytochrome c to the heme group involving 13 covalent bonds and one hydrogen bond (Fig. 1). A single cysteine residue was introduced at position 39 of yeast C102T iso-1-cytochrome c by site-directed mutagenesis, and labeled with a sulfhydryl-selective trisbipyridineruthenium reagent (26) to form Ru-39-cyt c (Fig. 1). This new derivative satisfied both design criteria listed above, and was used to measure the rate constant for intracomplex electron transfer from cytochrome c to Cu(A). The reaction was also studied as a function of ionic strength in order to provide information about the mechanism under physiological conditions.


Figure 1: Pathway for electron transfer in Ru-39-cyt c. The x-ray crystal structure of oxidized yeast iso-1-cytochrome c was obtained from the Brookhaven protein data base (File 2Ycyt c). His was substituted with a Cys and the ruthenium complex was attached to the sulfur atom of Cys. The proposed pathway for electron transfer extends from Ru-39 through the peptide backbone of Ser and Gly where there is a hydrogen bond from the amide nitrogen of Gly to the heme propionate group.




EXPERIMENTAL PROCEDURES

Materials

Beef heart cytochrome c oxidase was prepared as described by Capaldi and Hayashi (32) and had a heme content of 9-11 nmol/mg of protein and a turnover number of 400 s. The H39C,C102T variant of yeast iso-1-cytochrome c was prepared using site-directed mutagenesis as described by Hilgen and Pielak(33) . Wild-type yeast iso-1-cytochrome c has a free cysteine at position 102 that causes dimerization. Eighty seven percent of cytochromes c whose sequences are known have a threonine at this position(34) . Therefore, the C102T variant is used because it is more amenable to biophysical studies (35, 36, 37) and is structurally and functionally identical to the wild-type protein(30, 38, 39, 40) . It is also necessary to replace Cys-102 so it does not react with the ruthenium reagent. The H39C,C102T variant (530 µM) was treated with 260 µM dithiothreitol in 50 mM sodium borate, pH 9 for 15 min to reduce any disulfide cross-linked dimer, and then 1.8 mM (4-bromomethyl-4`-methylbipyridine) (bisbipyridine)ruthenium-(PF(6))(2) was added from a 105 mM stock solution in dry dimethylformamide(26) . After 16 h at room temperature, the reaction mixture was oxidized with 530 µM ferricyanide and passed through a 1 times 10-cm Bio-Gel P-2 column equilibrated with 10 mM sodium phosphate at pH 6 to remove excess reagent. The labeled protein was purified using a Waters 625 LC system equipped with a Waters 1 times 10-cm SP 8HR cation exchange column. The protein was eluted with a linear gradient from 10 to 500 mM sodium phosphate at pH 6 with a flow rate of 1 ml/min. A major peak eluting at 46 min (325 mM sodium phosphate) accounted for 87% of the total cytochrome, while a minor peak eluting at 42 min (300 mM) accounted for 13% (Fig. 2). The major peak was concentrated and washed twice with 5 mM sodium phosphate, pH 7, using an Amicon concentrator. The location of the ruthenium label was confirmed by digesting the derivative (1 mg/ml in 0.1 M Bicine, pH 8.0, 1% octyl glucoside) with two additions of 0.06 mg/ml tosylphenylalanyl chloromethyl ketone-treated trypsin for 15 h at 37 °C. The tryptic digest was chromatographed on a Dynamax 300 Å reverse-phase HPLC column with a linear gradient from 0.01% trifluoroacetic acid in water to 100% methanol and monitored using a Waters diode array detector. The ruthenium-labeled peptides were sequenced on an Applied Biosystems 473A sequenator.


Figure 2: Purification of Ru-39-cyt c. The crude reaction mixture of Ru-39-cyt c was eluted on a 1 times 10 cm Waters SP 8HR cation exchange column with a linear gradient from 10 to 500 mM sodium phosphate at pH 7 at a flow rate of 1 ml per min. The major peak eluting at 46 min (325 mM sodium phosphate) contained pure Ru-39-cyt c.



Flash Photolysis Experiments

Transient absorbance measurements of the rapid electron transfer between Ru(II) and the heme group in Ru-39-cyt c(^1)was carried out as described by Durham et al.(24) . Solutions containing 5-20 µM Ru-39-cyt c in 300 µM of 100 mM sodium phosphate, pH 7, were placed in semimicro glass cuvettes. The excitation pulse was provided by the third harmonic of a Nd:YAG laser, with a pulse width of 20 ns and a wavelength of 356 nm. The probe source was a pulsed 75 W xenon arc lamp, and the photomultiplier detector had a response time of 10 ns. The transient absorbance measurements for the reaction of Ru-39-cyt c with cytochrome c oxidase were carried out as described by Pan et al.(22) . The excitation source was a Phase R model DL 1400 flash lamp-pumped dye laser using coumarin 450 to produce a 450-nm light pulse of <0.5-µs duration. The photomultiplier detector had a response time of 1 µs. The reaction of cytochrome c was monitored at 550 nm using an extinction coefficient of Delta = 18.5 mM cm(41) . The reduction of heme a was measured at 604 nm using Delta = 16 mM cm and at 444 nm using Delta = 59 mM cm(42) . The reaction of Cu(A) was measured at 830 nm using Delta = 2.0 mM cm(43) . The extinction coefficients for the heme in Ru-39-cyt c at 604 and 830 nm were measured to be Delta = 1.2 mM cm and Delta = 0.15 mM cm. These values are less than 10% of the values of Delta for heme a and Delta for Cu(A), respectively, and therefore the small contributions of Ru-39-cyt c to the absorbance changes at 604 and 830 nm were ignored. The reaction solutions contained 5-10 µM Ru-39-cyt c, 5-20 µM cytochrome c oxidase, 10 mM aniline, 1 mM 3-carboxyl-2,2,5,5-tetramethyl-1-pyrolidinyloxy free radical (3CP), and 0.1% dodecyl maltoside in 5 mM sodium phosphate at pH 7.0 and 25 °C. The ionic strength was changed by adding sodium chloride. The reactions were carried out aerobically using resting state cytochrome c oxidase. Flash photolysis studies were also carried out using lumiflavin(21, 44) . The anaerobic solutions contained 100 µM lumiflavin, 5 mM EDTA, 4 µM cytochrome c, 0-5 µM cytochrome c oxidase in 0.1% dodecyl maltoside, 100 mM sodium phosphate buffer, pH 7.0. The transients were fitted to the appropriate theoretical equations as described by Durham et al.(24) and Pan et al.(22) , and the reported errors are the estimated standard deviations.


RESULTS

Preparation and Characterization of Ru-39-cyt c

The brominated ruthenium reagent was found to selectively label the single sulfhydryl group on the H39C,C102T yeast iso-1-cytochrome c variant. The yield of ruthenium-labeled protein was greater than 85%, with only minor amounts of other products. This reagent has been shown to be highly selective for labeling sulfhydryl groups under the conditions used, with very little labeling of other residues(26) . The tryptic digest of purified Ru-39-cyt c was found to contain a major ruthenium-labeled peptide with the sequence Ru-Cys-Ser-Gly-Gln-Ala-Glu-Gly-Tyr. A minor ruthenium-labeled peptide found in the tryptic digest was also labeled at Cys, but was cleaved at a different position. The purity of Ru-39-cyt c was determined to be greater than 95% from the HPLC chromatogram of the tryptic digest. The UV/visible spectra of Ru-39-cyt c was equal to the sum of the spectra of one equivalent of the ruthenium complex and one equivalent of native yeast iso-1-cytochrome c. There were no shifts in the positions of the absorption band maxima in either redox state. The 695-nm absorption band was retained, indicating that the bond between iron and the Met sulfur atom was intact. The redox potential measured as described by Geren et al.(26) is the same as for wild-type cytochrome c, 260 ± 10 mV. It was not possible to measure the redox potential of the ruthenium complex attached to the protein because of interference from the aqueous solvent. However, the emission maximum of Ru(II*) at 77 K is the same as that of the free complex, indicating that the redox potentials of the attached ruthenium complex are likely to be the same as those of the free complex, -0.85 V for the Ru(II*)/Ru(III) couple and +1.27 V for the Ru(II)/Ru(III) couple(45) .

Intraprotein Electron Transfer in Ru-39-cyt c

The luminescence emission spectrum of Ru-39-cyt c is centered at 618 nm, and has a decay rate of (6.0 ± 1.0) times 10^6 s in 0.1 M sodium phosphate at pH 7.0 and 25 °C. This is considerably larger than the value of 1.9 times 10^6 s for serum albumin labeled with the same ruthenium complex(26) , suggesting the possibility of oxidative quenching of Ru(II*) by the ferric heme. Transient absorption measurements using the techniques described by Durham et al.(24) were carried out to confirm this. Flash photolysis of a solution containing 12 µM Ru-39-cyt c in 100 mM sodium phosphate, pH 7, resulted in electron transfer from the photoexcited Ru(II*) to the heme Fe(III), followed by the thermal back reaction from Fe(II) to Ru(III) (Fig. S1). The reduction and reoxidation of the Ru-39-cyt c heme was measured at 550 nm. Ru(II*) makes a small contribution to the absorbance transient throughout the wavelength range from 530 to 570 nm (24) . The transient at 556.5 nm is due entirely to Ru(II*), since this is an isosbestic point for the heme group. The rate constant for this transient is 6 times 10^6 s which is identical to that of the luminescence decay. The heme Fe(II) transient was obtained by subtracting the 556.5-nm transient from the 550-nm transient, taking into account the slight difference in extinction coefficients at the two wavelengths (Fig. 3). The rise and fall times of the resulting Fe(II) transient were much slower than those of the 556.5 nm transient, consistent with electron transfer. The photoexcitation and recovery of Ru(II) was measured at 434 nm, an isosbestic for the heme group. Ru(II*) and Ru(III) do not contribute significantly to the absorbance at 434 nm. A unique set of values of k(1), k(2), and k(d) were required to simultaneously fit the 550-nm transient, the 434-nm transient, and the luminescence transient using the equations and procedures described by Durham et al.(24) . These values are k(1) = (0.6 ± 0.2) times 10^6 s, k(2) = (1.0 ± 0.2) times 10^6 s, and k(d) = (5.4 ± 0.6) times 10^6 s. These values are independent of protein concentration from 1-20 µM, indicating that they represent intraprotein electron transfer.


Scheme 1: Scheme 1




Figure 3: Photoinduced electron transfer within Ru-39-cyt c (12 µM in 100 mM sodium phosphate, pH 7). A, the Fe(II) transient was obtained from the difference between the 550-nm transient and the 556.5-nm transient as described in the text. B, 434-nm transient represents the photoexcitation and recovery of Ru(II). The smooth lines are the theoretical curves for Fe(II) (A) and Ru(II) (B) in Fig. S1, with k(1) = 6.0 times 10^5 s, k(2) = 1.0 times 10^6 s, and k = 5.4 times 10^6 s, using the equations given in Durham et al.(24) .



Intracomplex Electron Transfer between Ru-39-cyt c and Cytochrome c Oxidase

Laser flash photolysis of Ru-39-cyt c in the presence of the sacrificial donors aniline and 3CP resulted in a rapid increase in the 550 nm absorbance, with no subsequent decrease. This indicates rapid electron transfer from Ru(II*) to the heme Fe(III), followed by reduction of Ru(III) by aniline and 3CP to prevent the thermal back reaction k(2) (Fig. S1)(26) . When a solution containing Ru-39-cyt c and cytochrome c oxidase in low ionic strength buffer was excited with a laser flash, the heme was rapidly photoreduced to Fe(II), and then oxidized in a biphasic transient with rate constants of 6 times 10^4 s and 5 times 10^3 s, and amplitudes of 0.0018 and 0.0008 AU, respectively (Fig. 4A). The 830-nm absorbance first decreased with a rate constant of 6 times 10^4 s and then returned to its original value with a rate constant of 1.6 times 10^4 s, indicating reduction and reoxidation of Cu(A) (Fig. 4B). The 604-nm transient indicated that heme a was reduced with a rate constant of 1.8 times 10^4 s (Fig. 4C). The rate constants were independent of the concentration of Ru-39-cyt c and cytochrome c oxidase over the range 5-20 µM provided that the oxidase concentration was equal to or greater than that of Ru-39-cyt c. These results are consistent with intracomplex electron transfer according to :


Figure 4: Photoinduced electron transfer from Ru-39-cyt c to cytochrome c oxidase. The solution contained 12 µM Ru-39-cyt c and 18 µM cytochrome c oxidase in 5 mM sodium phosphate, pH 7, 10 mM aniline, 1 mM 3CP. A, 550-nm transient; B, 830-nm transient; C, 604-nm transient. The solid lines are the best fits to described in the text.



where c represents the oxidized form of Ru-39-cyt c present before the laser pulse, and (Cu(A)a) represents the resting form of cytochrome c oxidase. The transients at the three wavelengths were fit to the complete kinetic equations for , assuming k(1) k(a):

The fast phase of the reoxidation of Ru-39-cyt c had k(a) = (6 ± 2) times 10^4 s, and the amount of heme c reoxidized was 0.09 ± 0.01 µM. The 830-nm transient had k(a) = (6 ± 2) times 10^4 s, k(b) = (1.6 ± 0.3) times 10^4 s, and the amount of Cu(A) reduced and reoxidized was 0.11 ± 0.02 µM. The 604 nm transient had k(a) = (7 ± 3) times 10^4 s, k(b) = (1.8 ± 0.3) times 10^4 s and the amount of heme a reduced was 0.10 ± 0.01 µM. The transients at the three wavelengths are thus consistent with , with k(a) = (6 ± 2) times 10^4 s and k(b) = (1.8 ± 0.3) times 10^4 s. The transients indicate that each of the reactions in proceed in the forward direction with equilibrium constants of at least 5. The small slow phase in the 550-nm transient could be due to a minor form of the complex that is not properly aligned for rapid electron transfer from heme c to cytochrome c oxidase.

Ionic Strength Dependence of Reaction between Ru-39-cyt c and Cytochrome Oxidase

The effect of ionic strength on the kinetics of the reaction between Ru-39-cyt c and cytochrome c oxidase was determined in order to explore the mechanism under both intracomplex and bimolecular conditions. The rate constants and amplitudes of the 550-, 604-, and 830-nm transients for solutions containing 12.3 µM Ru-39-cyt c and 18 µM cytochrome c remained unchanged as the ionic strength was increased from 5 mM to 40 mM (Table 1). As the ionic strength was increased from 40 mM to 105 mM, the amplitudes of the fast phases of the transients at all three wavelengths progressively decreased to the same extent, but the rate constants k(a) and k(b) did not change (Table 1). The decrease in the amplitude of the fast phase was accompanied by the appearance of a slow phase with the same rate constant in the 550- and 604-nm transients, which increased in amplitude as the ionic strength was increased from 40 to 105 mM (Table 1). No corresponding slow phase was observed in the 830-nm transient. The rate constant of the slow phase increased linearly with the concentration of cytochrome c oxidase at 105 mM ionic strength (Fig. 5). This indicates that the slow phase is due to a bimolecular reaction between the two proteins, and a second-order rate constant of k = (1.7 ± 0.2) times 10^8M s was determined from the slope of the line in Fig. 5. The fraction of the fast phase increased from zero at low cytochrome c oxidase concentrations to 0.27 at 18 µM (Table 1). At ionic strengths above 105 mM no fast phases were observed in the 550- and 604-nm transients, and no transient at all was observed at 830 nm. The second-order rate constant measured from the concentration dependence of the slow phase decreased as the ionic strength was increased from 75 mM to 240 mM (Fig. 6).




Figure 5: Bimolecular electron transfer from Ru-39-cyt c to cytochrome c oxidase. The solutions contained 5 µM Ru-39-cyt c, 0 to 7 µM cytochrome c oxidase, 2 mM sodium phosphate, pH 7, 10 mM aniline, 1 mM 3CP, and 100 mM NaCl. The pseudo first-order rate constant k, in s, obtained from the 550- and 604-nm transients is plotted as a function of the concentration of cytochrome c oxidase in µM. The solid line is the best fit to a second-order rate constant of 1.75 times 10^8M s.




Figure 6: Dependence of the second-order rate constant of the reaction between Ru-39-cyt c and cytochrome c oxidase on ionic strength. The solutions contained 1-12 µM Ru-39-cyt c, 0-20 µM cytochrome c oxidase, 2 mM sodium phosphate, pH 7, 10 mM anline 1 mM 3CP, and 70-240 mM NaCl. The second-order rate constants, in units of M s, were measured as described in Fig. 5and are plotted as a function of the square root of the ionic strength, in units of M.



The kinetic results over the entire ionic strength range are consistent with the mechanism shown in :

At low ionic strength, all of the Ru-39-cyt c will be complexed with cytochrome c oxidase, and the reaction will proceed according to the top line of . At intermediate ionic strength (55-105 mM), there will be an equilibrium between complexed and uncomplexed Ru-39-cyt c represented by the dissociation constant K(d). The complexed Ru-39-cyt c will react according to the top line of , while the uncomplexed, photoreduced Ru-39-cyt c will first have to bind to cytochrome c oxidase and then transfer an electron. At ionic strengths above 100 mM, all of the Ru-39-cyt c will be uncomplexed before the laser flash, and will have to bind to cytochrome c oxidase before electron transfer can occur. The dissociation constant K(d) of the complex between Ru-39-cyt c and cytochrome c oxidase was determined from the fraction f of the fast phase of the 604-nm transients using :

where E(0) is the total concentration of cytochrome c oxidase and C(0) is the total concentration of Ru-39-cyt c. is based on a 1:1 stoichiometry for complex formation. The value of K(d) increased from less than 1 µM at low ionic strength to 8 µM at 75 mM ionic strength, and to greater than 100 µM at ionic strengths above 105 mM (Table 1). It was only possible to determine actual values for K(d) at ionic strengths between 55 and 105 mM where both the slow phase and the fast phase could be observed simultaneously.

The flavin flash photolysis method of Hazzard et al.(21) was used to compare the second-order rate constant of Ru-39-cyt c to that of wild-type iso-1-cytochrome c and horse cytochrome c at high ionic strength (100 mM sodium phosphate, pH 7, 5 mM EDTA). The second-order rate constants are the following: Ru-39-cyt c, (2.5 ± 0.4) times 10^7M s; wild-type yeast iso-1-cytochrome c, (1.7 ± 0.4) times 10^7M s; horse cytochrome c, (8.3 ± 2) times 10^6M s. The rate constant of Ru-39-cyt c is thus slightly larger than that of wild-type iso-1-cytochrome c. The same rate constant was obtained for Ru-39-cyt c using either lumiflavin excitation or ruthenium excitation.


DISCUSSION

Design Criteria for Ru-39-cyt c and Electron Transfer between Ruthenium and Heme

The design of Ru-39-cyt c was based on the identification of an efficient pathway for electron transfer between the ruthenium complex and the heme group which involves 13 covalent bonds and one hydrogen bond between the bipyridine group of the ruthenium complex and the porphyrin ring (31, 46) (Fig. 1). There are no through-space jumps and the single hydrogen bond bridges the amide of Gly and the heme propionate. The rate constants for electron transfer from Ru(II*) to Fe(III) and for the thermal back reaction from Fe(II) to Ru(III) are k(1) = 6 times 10^5 s and k(2) = 1.0 times 10^6 s. These rate constants should both be very close to the maximum, activationless rate constant for this system, since the driving forces of the two reactions (1.1 and 1.0 eV, respectively) are close to the expected reorganization energy of 0.8 eV (24) . The electron transfer rate constants of Ru-39-cyt c are comparable to the values k(1) = 1.4 times 10^6 s and k(2) = 3.2 times 10^6 s reported by Wuttke et al.(47) for Candida krusei cytochrome c modified at His-39 with Ru(bipyridine)(2)(imidazole). The driving force for the k(1) and k(2) reactions in this system are 1.2 and 0.74 eV, respectively. It should be noted that there are two fewer covalent bonds in the electron transfer pathway in the histidine-linked derivative than in Ru-39-cyt c, which is predicted to lead to a 3-fold decrease in the rate constant of the latter(31) . Taking this correction into account, the rate constants of the two derivatives are nearly identical. The Ru(bipyridine)(3) complex in Ru-39-cyt c is better suited for photoreduction experiments than the Ru(bipyridine)(2)(imidazole)(2) complex, because the excited state lifetime is much longer, and the Ru(III) state is more readily reduced with sacrificial donors(48) .

The location of the ruthenium group on the backside of Ru-39-cyt c was designed to allow normal interaction with cytochrome c oxidase. The second-order rate constant for the reaction of Ru-39-cyt c with cytochrome c oxidase at high ionic strength is nearly the same as that of wild-type iso-1-cytochrome c. This is one of the most important criteria for the functional integrity of the derivative. In addition, the redox potential and visible spectra, including the 695-nm band, are the same as for wild-type cytochrome c, indicating that the conformation of the heme crevice is not altered.

Intracomplex Electron Transfer between Ru-39-cyt c and Cytochrome c Oxidase

It has not been previously possible to measure the actual rate of electron transfer from cytochrome c to the initial acceptor in cytochrome c oxidase (20, 21, 22) . The present studies demonstrate that Ru-39-cyt c initially transfers an electron from heme c to Cu(A) with rate constant k(a) = 6 times 10^4 s, followed by electron transfer from Cu(A) to heme a with rate constant k(b) = 1.8 times 10^4 s (). This measurement is possible because the rate of reduction of heme c by Ru(II*) is much faster than the subsequent rate of electron transfer from heme c to Cu(A). The observed sequence of electron transfer, heme c Cu(A) heme a, indicates that the redox potentials for the three redox centers should increase in the same order. Although the redox potential of horse cytochrome c is 260 mV in solution, it decreases to 220 mV upon binding to cytochrome c oxidase(49) . The redox potential of Cu(A) has been reported to be 245 mV(50, 51) , while the redox potential of heme a is 362 mV when heme a(3) is oxidized(52) . Although these redox potentials are probably sensitive to the specific conditions used, the reported values are consistent with the sequence of electron transfer observed in the present experiments. The rate constant for electron transfer from Cu(A) to heme a, k(b) = 1.8 times 10^4 s, is the same as previously measured in fully oxidized cytochrome c oxidase (22, 53, 54) and in three-electron-reduced-CO-inhibited cytochrome c oxidase (55) . The reoxidation of heme a by the binuclear center is very slow in the present experiments, in agreement with previous studies utilizing resting cytochrome c oxidase(21, 22, 54, 56) .

Ionic Strength Dependence of Reaction between Ru-39-cyt c and Cytochrome c Oxidase, and Relevance to the Reaction under Physiological Conditions

The reactions of cytochrome c with its redox partners in intact mitochondria occur under physiological conditions of 100-150 mM ionic strength, and cytochrome c concentrations of 100-700 µM(57, 58) . Gupte and Hackenbrock (58) have proposed that under these conditions cytochrome c reacts by a three-dimensional diffusion process in the intermembrane space, with relatively little cytochrome c undergoing two-dimensional diffusion on the surface of the inner mitochondrial membrane. The reaction between cytochrome c and cytochrome c oxidase can be described by the following steps: 1) two- and three-dimensional diffusion of ferrocytochrome c to cytochrome c oxidase followed by formation of a transient substrate complex, 2) electron transfer within the substrate complex to form a product complex, and 3) dissociation of the product complex to release ferricytochrome c. Extensive studies under steady-state conditions have revealed the complexity of the overall reaction, and provided insight into certain aspects of the mechanism (for recent reviews, see Garber and Margoliash (59) and Cooper(60) ). To complement the information available from steady-state kinetics, we have used the ruthenium photoreduction technique to study the reaction as the conditions are changed continuously from low ionic strength where intracomplex kinetics are observed, to high ionic strength where bimolecular kinetics are observed.

The kinetics of the photoinduced reaction between Ru-39-cyt c and cytochrome c oxidase remain unchanged as the ionic strength is increased from 5 to 40 mM, and the equilibrium dissociation constant K(d) of the c:(Cu(A)a) complex remains less than 1 µM (Table 1). The reaction observed in the present experiments can therefore be correlated with the high affinity phase of the steady-state reaction, which has a K(m) of 9 times 10M for native yeast iso-1-cytochrome c in 25 mM ionic strength buffer at pH 7.8(15, 61) . The K(m) value determined from the steady-state kinetics is essentially the same as the equilibrium dissociation constant K(d) determined from direct binding studies of the oxidized proteins(61) . Furthermore, the turnover number for the high affinity phase has been shown to be rate-limited by the dissociation of the product complex at low ionic strength(62, 63) .

In some respects the most revealing results of the ruthenium photoreduction experiments are observed at intermediate ionic strength (55-105 mM), where both intracomplex and bimolecular kinetics are present simultaneously (Table 1). The fact that the rate constant for the fast phase of electron transfer from c to Cu(A) does not change as the ionic strength increases from 5 to 105 mM indicates that the dissociation rate constant k is much smaller that k(a) (). If k were larger than k(a), then rapid equilibrium conditions would apply, and separate slow and fast phases would not be observed. Assuming that the bimolecular reaction proceeds according to , the second-order rate constant determined from the slow phase will be given by :

is based on the steady-state assumption, which should be valid in the present case because the concentration of cytochrome c oxidase is much larger than the concentration of photoreduced Ru-39-cyt c. Since k is much smaller than k(a) at ionic strengths up to 105 mM, the second-order rate constant will be equal to k under these conditions. The value of k is thus 2.2 times 10^8M s at 75 mM ionic strength, and 1.7 times 10^8M s at 105 mM ionic strength. If it is assumed that the equilibrium dissociation constant K(d) measured for the c:(Cu(A)a) complex is the same for the c:(Cu(A)a) complex, then K(d) = k/k. This is a reasonable assumption, since the Minnaert mechanism IV that accounts for the first-order time course of the steady-state reaction is based on equal dissociation constants for oxidized and reduced cytochrome c(64, 65) . With this assumption, the values of k are 1.4 times 10^3 s at 75 mM ionic strength, and 8.5 times 10^3 s at 105 mM ionic strength, which are significantly less than k(a).

At the lower limit of physiological ionic strength, 105 mM, the reaction can be described by complex formation with rate constant k = 1.7 times 10^8M s, intracomplex electron transfer with rate constant k(a) = 6 times 10^4 s, and complex dissociation with rate constant k = 8.5 times 10^3 s. Since k is small compared to k(a), essentially all of the ferrocytochrome c that binds to the high affinity site will react before dissociation. As the ionic strength is increased above 105 mM, k will increase and k will decrease. At some ionic strength it is likely that k will become comparable to k(a), and k will be a function of all three rate constants as given in . Since k(a) is unchanged over the ionic strength range from 5 to 105 mM, it will probably remain unchanged as the ionic strength is increased further. The decrease in k with increasing ionic strength is thus probably due to an increase in k and/or a decrease in k rather than to changes in k(a). Ionic strength dependence studies have been used extensively to characterize electrostatic interactions between redox proteins, and it is generally agreed that the slope of a plot of log k versus the square root of the ionic strength is proportional to the strength of the electrostatic interaction(14, 66, 67) . The slope of the plot for k shown in Fig. 6is essentially the same as that of a plot of the steady-state parameter V(max)/K(m) for the reaction between horse cytochrome c and beef cytochrome c oxidase(14) . This indicates that the electrostatic interactions are similar for yeast Ru-39-cyt c and native horse cytochrome c.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants GM20488 (to F. M. and B. D.) and GM42501 (to G. J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Partially supported by National Institutes of Health Protein Engineering and Molecular Genetics Training Grant GM08332.

Partially supported by the Department of Biochemistry and Biophysics.

**
To whom correspondence should be addressed. Tel.: 501-575-4999; Fax: 501-575-4049.

(^1)
The abbreviations used are: Ru-39, (dimethylbipyridine) (bisbipyridine)ruthenium-Cys-39; cyt c, cytochrome c; 3CP, 3-carboxyl-2,2,5,5-tetramethyl-1-pyrolidinyloxy free radical; HPLC, high performance liquid chromatography; Bicine, N,N-bis-(2-hydroxyethyl)glycine.


ACKNOWLEDGEMENTS

We thank Chuan Chen for help with some of the kinetic studies.


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