Formation of electrostatically-stabilized complex at low ionic strength inhibits interprotein electron transfer between yeast cytochrome c and cytochrome c peroxidase

Electron transfer from yeast ferrous cytochrome c to H2O2-oxidized yeast cytochrome c peroxidase has been studied using flash photoreduction methods. At low ionic strength (mu less than 10 mM), where a strong complex is formed between cytochrome c and peroxidase, electron transfer occurs rather slowly (k approximately 200s-1). However, at high ionic strength where the electrostatic complex is largely dissociated, the observed first-order rate constant for peroxidase reduction increases significantly reaching a concentration independent limit of k approximately 1500 s-1. Thus, at least in some cases, formation of an electrostatically-stabilized complex can actually impede electron transfer between proteins.     

Kinetics of intracomplex electron transfer and of reduction of the components of covalent and noncovalent complexes of cytochrome c and cytochrome c peroxidase by free flavin semiquinones

The kinetics of reduction of free flavin semiquinones of the individual components of 1:1 covalent and electrostatic complexes of yeast ferric and ferryl cytochrome c peroxidase and ferric horse cytochrome c have been studied. Covalent cross-linking between the peroxidase and cytochrome c at low ionic strength results in a complex that has kinetic properties both similar to and different from those of the electrostatic complex. Whereas the cytochrome c heme exposure to exogenous reductants is similar in both complexes, the apparent electrostatic environment near the cytochrome c heme edge is markedly different. In the electrostatic complex, a net positive charge is present, whereas in the covalent complex, an essentially neutral electrostatic charge is found. Intracomplex electron transfer within the two complexes is also different. For the covalent complex, electron transfer from ferrous cytochrome c to the ferryl peroxidase has a rate constant of 1560 s-1, which is invariant with respect to changes in the ionic strength. The rate constant for intracomplex electron transfer within the electrostatic complex is highly ionic strength dependent. At mu = 8 mM a value of 750 s-1 has been obtained [Hazzard, J. T., Poulos, T. L., & Tollin, G. (1987) Biochemistry 26, 2836-2848], whereas at mu = 30 mM the value is 3300 s-1. This ionic strength dependency for the electrostatic complex has been interpreted in terms of the rearrangement of the two proteins comprising the complex to a more favorable orientation for electron transfer. In the case of the covalent complex, such reorientation is apparently impeded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis.

The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.     

Photoinduced electron transfer between cytochrome c peroxidase and horse cytochrome c labeled at specific lysines with (dicarboxybipyridine)(bisbipyridine)ruthenium(II)

The reactions of yeast cytochrome c peroxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) [Ru(II)] were studied by flash photolysis. All of the derivatives formed complexes with cytochrome c peroxidase compound I (CMPI) at low ionic strength (2 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in electron transfer to the ferric heme group in cytochrome c, followed by electron transfer to the radical site in CMPI. This reaction was biphasic and the rate constants were independent of CMPI concentration, indicating that both phases represented intracomplex electron transfer from the cytochrome c heme to the radical site in CMPI. The rate constants of the fast phase were 5200, 19,000, 55,000, and 14,300 s-1 for the derivatives modified at lysines 13, 25, 27, and 72, respectively. The rate constants of the slow phase were 260, 520, 200, and 350 s-1 for the same derivatives. These results suggest that there are two binding orientations for cytochrome c on CMPI. The binding orientation responsible for the fast phase involves a geometry that supports rapid electron transfer, while that for the slow phase allows only slow electron transfer. Increasing the ionic strength up to 40 mM increased the rate constant of the slow phase and decreased that of the fast phase. A single intracomplex electron transfer phase with a rate constant of 2800 s-1 was observed for the lysine 72 derivative at this ionic strength. When a series of light flashes was used to titrate CMPI to CMPII, the reaction between the cytochrome c derivative and the Fe(IV) site in CMPII was observed. The rate constants for this reaction were 110, 250, 350, and 140 s-1 for the above derivatives measured in low ionic strength buffer.     

Chromatium flavocytochrome c: kinetics of reduction of the heme subunit, and the flavocytochrome c-mitochondrial cytochrome c complex

The kinetics of reduction of Chromatium vinosum flavocytochrome c heme subunit by exogenous flavin neutral semiquinones generated by laser flash photolysis have been investigated. Unlike the holoprotein, the isolated heme subunit was appreciably reactive with lumiflavin neutral semiquinone. The measured rate constant for the reaction (2.7 X 10(7) M-1 S-1) was comparable to those of c-type cytochromes having similar redox potentials. The ionic strength dependence of the reaction with FMN neutral radical indicated that the heme subunit had a small negative charge at the site of reduction. Taken together, these results suggest that the active site of the heme subunit is buried on complexation with the flavin subunit in the holoprotein. Horse cytochrome c formed a strong complex with Chromatium, but not Chlorobium, flavocytochrome c. Possible physiological electron acceptors such as HiPIP, cytochrome c', and cytochrome c-555 apparently did not bind to the flavocytochromes c. The rate constant for reduction by lumiflavin radical of horse cytochrome c complexed to flavocytochrome c was about twofold smaller than for reduction of horse cytochrome c alone. Flavocytochrome c was itself unreactive with exogenous flavin semiquinones. The ionic strength dependence of the reduction of the complex by FMN radical was also smaller than for horse cytochrome c in the absence of flavocytochrome c. Sulfite, which forms an adduct with the protein-bound FAD (FAD is bound in an 8-alpha-S-cysteinyl linkage), did not affect the reduction of horse cytochrome c in its complex with flavocytochrome c. We conclude that horse cytochrome c is reduced directly by exogenous flavins in its complex with flavocytochrome c, although the kinetics are slightly modified. These results are not unlike observations made with complexes of mitochondrial cytochrome c with cytochrome oxidase or cytochrome b5.     

In vitro kinetics of reduction of cytochrome c554 isolated from the reaction center of the green phototrophic bacterium, Chloroflexus aurantiacus

The photochemical reaction center in the green bacterium Chloroflexus aurantiacus is similar to that found in purple phototrophic bacteria and interacts with a multiheme membrane-bound cytochrome. We have examined the kinetics of reduction of the pure solubilized reaction center cytochrome by laser flash photolysis of solutions containing lumiflavin or FMN. Reduction by lumiflavin semiquinone followed single exponential kinetics and the observed rate constant (kobs) was linearly dependent on protein concentration (k = 1.8 X 10(7) M-1s-1 heme-1). This result suggests either that the four hemes have similar reduction rate constants which cannot be resolved or that there are large differences in rate constant and only the most reactive heme (or hemes) was observed under these conditions. To determine the relative reactivities of the four hemes, we varied the extent of heme reduction at a single total protein concentration. As the hemes were progressively reduced by steady-state illumination prior to laser flash photolysis, kobs for the reaction with fully reduced lumiflavin decreased nonlinearly. Second-order rate constants for the four hemes were assigned by nonlinear least-squares analysis of kobs vs oxidized heme concentration data. The second-order rate constants obtained in this way for the highest and lowest potential hemes differed by a factor of about 20, which is larger than expected for c-type cytochromes based on redox potential alone (a factor of about 3 would be expected). This is interpreted as being due to differences in steric accessibility. Relative to the highest potential heme, which is as reactive as a typical c-type cytochrome, we estimated a steric effect of approximately twofold for heme 2, and steric effects of approximately fivefold for hemes 3 and 4. Using fully reduced FMN as reductant of oxidized cytochrome, ionic strength effects indicate a minus-minus interaction, with approximately a -2 charge near the site of reduction of the highest potential heme.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with Ki values of 4.5, 91, 200 and 330 microM, respectively. 2. The inhibition constant Ki of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme. 3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k1 of 33 M-1 . s-1 and a dissociation constant Kd of 3.9 mM. 4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M-1 . s-1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes. 5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome alpha 3-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea.

During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction kinetics of the ferredoxin-ferredoxin-NADP+ reductase complex: a laser flash photolysis study

The kinetics of reduction of spinach ferredoxin (Fd), ferredoxin-NADP+ reductase (FNR), and the Fd-FNR complex have been investigated by the laser flash photolysis technique. 5-Deazariboflavin semiquinone (5-dRf), generated in situ by laser flash photolysis under anaerobic conditions, rapidly reduced both oxidized Fd (Fdox) (k = 2 X 10(8) M-1 s-1) and oxidized FNR (FNRox) (K = 6.3 X 10(8) M-1 s-1) at low ionic strength (10 mM) at pH 7.0, leading to the formation of reduced Fd (Fdred) and FNR semiquinone (FNR.), respectively. At higher ionic strengths (310 and 460 mM), the rate constant for the reduction of the free Fdox increased about 3-fold (k = 6.7 X 10(8) M-1 s-1 at 310 mM and 6.4 X 10(8) M-1 s-1 at 460 mM). No change in the second-order rate constant for reduction of the free FNRox was observed at high ionic strength. At low ionic strength (10 mM), 5-dRf. reacted only with the FAD center of the preformed 1:1 Fdox-FNRox complex (k = 5.6 X 10(8) M-1 s-1), leading to the formation of FNR.. No direct reduction of Fdox in the complex was observed. No change in the kinetics occurred in the presence of excess NADP+. The second-order rate constant for reduction of Fdox by 5-dRf. in the presence of a stoichiometric amount of fully reduced FNR at low ionic strength was 7 X 10(6) M-1 s-1, i.e., about one-thirtieth the rate constant for reduction of free Fdox.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new ruthenium complex to study single-electron reduction of the pulsed O(H) state of detergent-solubilized cytochrome oxidase

The first step in the catalytic cycle of cytochrome oxidase, the one-electron reduction of the fully oxidized enzyme, was investigated using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine)2]2(quaterpyridine), (Ru2Z). The aim of the work was to examine differences in the redox kinetics resulting from pulsing the oxidase (i.e., fully reducing the enzyme followed by reoxidation) just prior to photoreduction. Recent reports indicate transient changes in the redox behavior of the metal centers upon pulsing. The new photoreductant has a large quantum yield, allowing the kinetics data to be acquired in a single flash. The net charge of +4 on Ru2Z allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. The photoexcited state Ru(II*) of Ru2Z is reduced to Ru(I) by the sacrificial electron donor aniline, and Ru(I) then reduces CuA with yields up to 60%. A stopped-flow-flash technique was used to form the pulsed state of cytochrome oxidase (the "OH" state) from several sources (bovine heart mitochondria, Rhodobacter sphaeroides, and Paracoccus denitrificans). Upon mixing the fully reduced anaerobic enzyme with oxygenated buffer containing Ru2Z, the oxidized OH state was formed within 5 ms. Ru2Z was then excited with a laser flash to inject one electron into CuA. Electron transfer from CuA --> heme a --> heme a3/CuB was monitored by optical spectroscopy, and the results were compared with the enzyme that had not been pulsed to the OH state. Pulsing had a significant effect in the case of the bovine oxidase, but this was not observed with the bacterial oxidases. Electron transfer from CuA to heme a occurred with a rate constant of 20,000 s-1 with the bovine cytochrome oxidase, regardless of whether the enzyme had been pulsed. However, electron transfer from heme a to the heme a3/CuB center in the pulsed form was 63% complete and occurred with biphasic kinetics with rate constants of 750 s-1 and 110 s-1 and relative amplitudes of 25% and 75%. In contrast, one-electron injection into the nonpulsed O form of the bovine oxidase was only 30% complete and occurred with monophasic kinetics with a rate constant of 90 s-1. This is the first indication of a difference between the fast form of the bovine oxidase and the pulsed OH form. No reduction of heme a3 is observed, indicating that CuB is the initial electron acceptor in the one-electron reduced pulsed bovine oxidase.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis.

The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser flash photolysis studies of the reduction kinetics of NADPH:cytochrome P-450 reductase

The reduction kinetics of NADPH:cytochrome P-450 reductase have been investigated by the laser flash photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Transients observed at 470 nm at neutral pH indicated that the oxidized reductase was reduced via second-order kinetics with a rate constant of 6.8 X 10(7) M-1 s-1. The second-order rate constant corresponding to the formation of the protein-bound semiquinone (measured at 585 nm) was essentially the same as that obtained at 470 nm (7.1 X 10(7) M-1 s-1). Subsequent to this rapid formation of protein-bound semiquinone, a partial exponential decay was observed at 585 nm. The rate of this decay remained invariant with protein concentration between pH 5.0 and 7.0, and a first-order rate constant of 70 s-1 was obtained for this process. This is assigned to intramolecular electron transfer from FADH. to FMN. Prior reduction of the enzyme to the one-electron level led to a decrease in both the second-order rate constant for reduction (2 X 10(7) M-1 s-1) and the first-order intraflavin electron transfer rate constant (15 s-1). The protein-bound FAD moiety of FMN-depleted reductase was reduced by 5-dRfH. with a second-order rate constant that was identical with that observed with the native enzyme (6.9 X 10(7) M-1 s-1). However, with this species no significant decay of the FAD semiquinone was observed at 585 nm following its rapid formation, consistent with the above assignment of this kinetic process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser flash photolysis studies of electron transfer between semiquinone and fully reduced free flavins and the cytochrome c-cytochrome oxidase complex

Laser flash photolysis has been used to determine the rate constants for the reduction of bovine cytochrome oxidase and the cytochrome c-cytochrome oxidase complex by the semiquinone and fully reduced forms of various flavin analogues (FH. and FH-, respectively). Under the condition used, the reaction of FH. with free cytochrome oxidase is too slow to compete with FH. disproportionation whereas FH- reacts measurably. Both FH. and FH- are effective in reducing the complex. The reduction of heme a in the complex is shown to proceed via cytochrome c, and a limiting first-order rate is observed in the case of FH- at high complex concentrations. The data indicate that the interaction site for electron transfer to cytochrome c is the same in the complex as with the free protein, and although a tight complex exists, at least small reactants like the flavins are not sterically hindered in their access to the bound cytochrome c. Moreover, the results also establish that intramolecular electron transfer between cytochrome c and cytochrome oxidase within the complex occurs with a first-order rate constant of greater than 700 s-1. Thus, the presence of cytochrome c greatly enhances electron transfer from reduced flavins to cytochrome oxidase.     

Solvent proton relaxation studies of cytochrome c oxidase solutions

The interaction of solvent water protons with the bound paramagnetic metal ions of beef heart cytochrome c oxidase has been examined. The observed proton relaxation rates of enzyme solutions had a negative temperature dependence, indicating a rapid exchange between solvent protons in the coordination sphere of the metal ions and bulk solvent. An analysis of the dependence of the proton relaxation rate on the observation frequency indicated that the correlation time, which modulates the interaction between solvent protons and the unpaired electrons on the metal ions, is due to the electron spin relaxation time of the heme irons of cytochrome c oxidase. This means that at least one of the hemes is exposed to solvent. The proton relaxation rate of the oxidized enzyme was found to be sensitive to changes in ionic strength and to changes in the spin states of the metal ions. Heme a3 was found to be relatively inaccessible to bulk solvent. Partial reduction of the enzyme caused a slight increase in the relaxation rate, which may be due to a change in the antiferromagnetic coupling between two of the bound paramagnetic centers. Further reduction resulted in a decreased relaxation rate, and the fully reduced enzyme was no longer sensitive to changes in ionic strength. The binding of cytochrome c to cytochrome c oxidase had little effect on the proton relaxation rates of oxidized cytochrome oxidase indicating that cytochrome c binding has little effect on solvent accessibility to the metal ion sites.     

Effect of changing the detergent bound to bovine cytochrome c oxidase upon its individual electron-transfer steps

The influence of the detergent environment upon individual electron-transfer rates of cytochrome c oxidase was investigated by stopped-flow spectrophotometry. The effects of three detergents were studied: lauryl maltoside, which supports a high turnover number (TN = 350 s-1), n-dodecyl octaethylene glycol monoether (C12E8), which supports an intermediate TN (150 s-1), and Triton X-100 in which oxidase is nearly inactive (TN = 2-3 s-1). Under limited turnover conditions (cytochrome c:cytochrome c oxidase ratio = 1:1 to 8:1), the rate of oxidation of cytochrome c was measured and compared with the fast reduction of cytochrome a and its relatively slow reoxidation. Two reducing equivalents of cytochrome c were rapidly oxidized in a burst phase; the remaining two to six equivalents were oxidized more slowly, concurrent with the reoxidation of cytochrome a; i.e., the percent reduced cytochrome a reflects the percent reduced cytochrome c. With the resting enzyme, the bimolecular reaction between reduced cytochrome c and cytochrome a was rapid, was insensitive to the detergent environment, and was not the rate-limiting step in the presence of any detergent. The rate of internal electron transfer from cytochrome a to cytochrome a3 in the resting enzyme was slow and only slightly affected by the detergent environment: 1.0-1.1 s-1 in Triton X-100, 5-7 s-1 in C12E8, and 5-12 s-1 in lauryl maltoside. With the pulsed enzyme, the intramolecular electron transfer between cytochrome a and cytochrome a3 increased 4-5-fold in the lauryl maltoside enzyme but did not increase in the Triton X-100 enzyme (intermediate values were obtained with the C12E8 enzyme). We conclude that cytochrome c oxidase acquires the pulsed conformation only in those detergents that support high TN's, e.g., lauryl maltoside and C12E8, but it is locked in the resting conformation in those detergents which result in low TN's, e.g., Triton X-100.     

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. I. The interaction of cytochrome c2 with the reaction center

1. The kinetics of the interaction of cytochrome c2 and photosynthetic reaction centers purified from Rhodobacter capsulatus were studied in proteoliposomes reconstituted with a mixture of phospholipids simulating the native membrane (i.e. containing 25% L-alpha-phosphatidylglycerol). 2. At low ionic strength, the kinetics of cytochrome-c2 oxidation induced by a single turnover flash was very different, depending on the concentration of cytochrome c2: at concentrations lower than 1 microM, the process was strictly bimolecular (second-order rate constant, k = 1.7 x 10(9) M-1 s-1), while at higher concentrations a fast oxidation process (half-time lower than 20 microseconds) became increasingly dominant and encompassed the total process at a cytochrome c2 concentration around 10 microM. From the concentration dependence of the amplitude of this fast phase an association constant for a reaction-center--cytochrome-c2 complex of about 10(5) M-1 was evaluated. From the fraction of photo-oxidized reaction centers promptly re-reduced in the presence of saturating concentrations of externally added cytochrome c2, it was found that in approximately 60% of the centers the cytochrome-c2 site was exposed to the external compartment. 3. Both the second-order oxidation reaction and the formation of the reaction-center--cytochrome-c2 complex were very sensitive to ionic strength. In the presence of 180 mM KCl, the value of the second-order rate constant was decreased to 7.0 x 10(7) M-1 s-1 and no fast oxidation of cytochrome c2 could be observed at 10 microM cytochrome c2. 4. The kinetics of exchange of oxidized cytochrome c2 bound to the reaction center with the reduced form of the same carrier, following a single turnover flash, was studied in double-flash experiments, varying the dark time between photoactivations over the range 30 microseconds to 5ms. The experimental results were analyzed according to aminimal kinetic model relating the amounts of oxidized cytochrome c2 and reaction centers observable after the second flash to the dark time between flashes. This model included the rate constants for the electron transfer between the primary and secondary ubiquinone acceptors of the complex (k1) and for the exchange of cytochrome c2 (k2). Fitting to the experimental results indicated a value of k1 equal to 2.4 x 10(3) s-1 and a lower limit for k2 of approximately 2 x 10(4) s-1 (corresponding to a second-order rate constant of approximately 3 x 10(9) M-1 s-1).     

Design of a ruthenium-labeled cytochrome c derivative to study electron transfer with the cytochrome bc1 complex

A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc1 to cytochrome c (Cc). The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru(z)-39-Cc. The Ru(z)-39-Cc derivative has the same steady-state activity with yeast cytochrome bc1 as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction. Laser excitation of reduced Ru(z)-39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10(6) x s(-1). The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer. The yield of photooxidized heme c is 20% in a single flash. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc1 and Ru(z)-39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome c1 to heme c with a rate constant of 1.4 x 10(4) x s(-1). As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc1. The interaction of yeast Ru-39-Cc with yeast cytochrome bc1 is stronger than that of horse Ru-39-Cc with bovine cytochrome bc1, suggesting that nonpolar interactions are stronger in the yeast system.     

Cytochrome c reduction by cysteine plus copper: a pseudosubstrate system for cytochrome c oxidase

Cysteine alone reduces horse heart cytochrome c very slowly (k approximately or equal too 1.0 M-1s-1) with a rate constant virtually identical in high and low ionic strength buffers. Copper catalyzes this reaction increasing the rate by a factor of 10(5) in 50 mM phosphate and by a factor of 10(6) in 10mM Tris buffers. When ferricytochrome c and cysteine are mixed in an oxygen electrode a "burst" of oxygen uptake is seen, the decline in which parallels the reduction of cytochrome c. When cytochrome oxidase is added to such a mixture two routes of electron transfer to oxygen exist: enzymatic and ferricytochrome c dependent nonenzymatic. Both processes are sensitive to cyanide, but azide inhibits only the authentic cytochrome c oxidase catalyzed process and BCS the ferricytochrome c stimulated reaction.     

Kinetic studies on oxidized and partially reduced cytochrome c oxidase.

1. Kinetic studies have been performed with beef-heart cytochrome c oxidase, with the enzyme either in its oxidized, resting state or pretreated anaerobically with different amounts of reduced cytochrome c. The techniques used for the study have been stopped-flow spectrophotometry and electron paramagnetic resonance (EPR) spectroscopy. 2. The results show that the one-electron equivalent-reduced enzyme rapidly oxidizes one further equivalent of aerobically or anaerobically added ferrocytochrome c, with a rate constant of 5 . 10(6) M-1 . s-1. 3. When an excess of ferrocytochrome c in the presence of oxygen is added to the one-electron-reduced enzyme, the same turnover rate is obtained as in experiments with the resting enzyme. 4. The one-electron equivalent-enzyme reacts with CO with a rate constant of 4 . 10(4) M-1 . s-1 to yield approx. 35% of the CO compound as compared with the reaction between the fully reduced enzyme and CO. 5. It is shown that on reduction the enzyme is converted into an active form, but it is concluded that the enzyme does not have to be fully reduced before it is catalytically active.