Single and multiple turnover reactions in the ubiquinone-cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroids: the physical chemistry of the major electron donor to cytochrome c2, and its coupled reactions.

We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c2 in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferricytochrome with a halftime of 1-2 ms under optimal conditions, has an oxidation-reduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction. The state of reduction of Z, which may be a quinone.protein complex near the inner (cytochrome c2) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c2, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome rereduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c2 oxido-reductase.     

Single and multiple turnover reactions in the ubiquinone-cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroides. The physical chemistry of the major electron donor to cytochrome c2, and its coupled reactions

We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c₂ in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferri-cytochrome with a halftime of 1–2 ms under optimal conditions, has an oxidation-reduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction.

The state of reduction of Z, which may be a quinone · protein complex near the inner (cytochrome c₂) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c₂, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome re-reduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c₂ oxido-reductase.     

The nature and magnitude of the charge-separation reactions of ubiquinol cytochrome c2 oxidoreductase

The transdielectric charge separation reaction catalyzed by the ubiquinol-cytochrome c2 oxidoreductase is achieved in two fractional steps. We present a detailed analysis which addresses the nature of the charge transferred, the redox groups directly involved in charge separation and the contributions of each to the full charge separation catalyzed by the enzyme. Accounting for light saturation effects, reaction centers unconnected to cytochrome c2 and the fraction of total cytochrome bc1 turning over per flash permits detailed quantitation of: (1) the red carotenoid bandshift associated with electron transfer between ubiquinol at site Qz and the high- (2Fe2S center, cytochrome c1) and low-potential (cytochrome bL, cytochrome bH) components of cytochrome bc1; (2) the blue bandshift accompanying reduction of cytochrome bH by ubiquinol via site Qc (the reverse of the physiological reaction); and (3) the effect of delta psi on the Qc-cytochrome bH redox equilibrium. Studies were performed at pH values above and below the redox-linked pK values of the redox centers known to be involved in each reaction at equilibrium. The conclusions of this study may be summarized as follows: (1) there is no transdielectric charge separation apparent in the redox reactions between Qz and cytochrome bL, 2Fe2S and cytochrome c1 (in agreement with Glaser, E. and Crofts, A.R. (1984) Biochim. Biophys. Acta 766, 223-235), i.e., charge separation accompanies electron transfer between cytochrome bL and cytochrome bH; (2) the redox reactions between cytochrome bL and cytochrome bH and between cytochrome bH and Qc constitute the full electrogenic span; (3) electron transfer between cytochrome bL and cytochrome bH contributes approx. 60% of this span; (4) electron transfer between cytochrome bH and Qc contributes 45-55% as calculated from the blue bandshift or the delta psi-dependent equilibrium shift; (5) there is no discernable pH dependence of the Qz-cytochrome bH or Qc-cytochrome bH charge-separation reactions; (6) cytochrome bL, Qz, 2Fe2S, and cytochrome c1 are on the periplasmic side out of the low dielectric part of the membrane while cytochrome bH is buried in the low dielectric medium; (7) electron transfer is the predominant if not the sole contributor to charge separation; (8) Qz and Qc are on opposite sides of the membrane dielectric profile.     

On the question of interheme electron transfer in the chloroplast cytochrome b6 in situ

The redox properties, the site of action of the inhibitor NQNO, and the question of interheme transfer in the chloroplast cytochrome b6 have been examined with regard to the role of the b6-f complex in quinol oxidation and H+ translocation. (i) The two hemes of the cytochrome ba and bp, have similar (delta Em less than or equal to 50 mV) oxidation-reduction midpoint potentials that are pH-independent in the range pH 6.5-8.0 (Em7 = -40 mV) but are pH dependent below this range with an estimated pK = 6.7. (ii) Only half of cytochrome b6, the stromal-side heme, ba, was reducible by NADPH and ferredoxin. (iii) The 2-3-fold increase (to 0.60 +/- 0.09 heme/600 Chl) in the amplitude of flash-induced cytochrome reduction caused by NQNO was not affected when heme ba was initially reduced, implying that NQNO affects flash reduction at the site of heme bp. (iv) Multiple light flashes did not increase the amplitude of b6 reduction in the presence or absence of NQNO or show binary oscillations. Together with localization of a site of action of NQNO near heme bp, these data provide no evidence for efficient electron transfer from heme bp to heme ba as specified by the Q cycle model. (v) NQNO interaction with heme bp does not block its oxidation, since reoxidation of the flash-reduced cytochrome in its presence or absence was 4-5 times faster (t1/2 approximately 30 ms) when heme ba was reduced. The faster oxidation of the photoreduced cytochrome after NADPH-Fd reduction of heme ba indicates that the oxidation of ba and bp may be cooperative.     

Inhibitory effect of N,N'-dicyclohexylcarbodiimide (DCCD) on the electron transfer involving cytochrome b-c2 oxidoreductase in Rhodopseudomonas sphaeroides

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibited dark re-reduction of cytochrome c2 and reduction of b-type cytochrome, both of which are closely associated with electron transfer involving a cytochrome b-c2 oxidoreductase, after a single-turnover flash excitation in the chromatophore membranes from a photosynthetic bacterium, Rhodopseudomonas sphaeroides. Rapid proton uptakes (HI+, HII+) and the formation of the membrane potential registered by carotenoid bandshift phase III were also inhibited by DCCD. The electron transfer was inhibited in the presence of either valinomycin or carbonylcyanide-m-chlorophenylhydrazone (CCCP). These results indicated that DCCD inhibited the electron transfer involving the cytochrome b-c2 oxidoreductase in the bacterium. The inhibition was irreversible. A hydrophilic carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), did not affect the above-mentioned reactions. Thus, DCCD may interact with the hydrophobic region(s) in the chromatophore membranes from photosynthetic bacteria resulting in the inhibition(s) of the photosynthetic cyclic electron transfer.     

Some properties of mammalian cardiac cytochrome c1.

Investigations into the nature of the axial heme ligands, the strength of the heme crevice, the reactivity with cyanide, and the ascorbate reducibility of cytochrome c1 were performed to explore structure-function relationships of cytochrome c1. The existence of an absorbance band at 690 nm, which was quenched by raising the pH with a pK of 9.2 corresponding to a low spin-low transition, suggested that a methionine residue probably functioned as one of the axial heme iron ligands in this cytochrome. Spectral titrations of cytochrome c1 in the low pH range showed a markedly elevated pK for the low spin-high spin transition relative to cytochrome c. Denaturation studies with urea, the absence of any reaction with cyanide, and the evidence from other lines would appear to indicate that the heme group of cytochrome c1 was reduced by ascorbate at approximately 5% of the rate of reduction of cytochrome c but this rate dramatically increased with increasing pH concomitant with the disappearance of the 690 nm absorbance band. Circular dichroic spectra substantiated that elevated pH produced conformational changes localized to the heme crevice and probably also the regions containing aromatic residues. The enhanced rate of ascorbate reduction was perhaps a consequence of the increased accessibility of the heme iron to ascorbate. Major unfolding of the protein in 8 M urea, however, completely abolished the ascorbate reducibility of cytochrome c1. The buried nature of the heme group of cytochrome c1 would probably preclude transfer of an electron from cytochrome c1 to cytochrome c through a direct Fe-Fe or a heme-heme interaction. This poses an important question concerning the mechanism of this electron transfer between these two cytochromes not only in mitochondria but also in solution.     

Electron transfer and stability of the cytochrome b6f complex in a small domain deletion mutant of cytochrome f

The lumen segment of cytochrome f consists of a small and a large domain. The role of the small domain in the biogenesis and stability of the cytochrome b(6)f complex and electron transfer through the cytochrome b(6)f complex was studied with a small domain deletion mutant in Chlamydomonas reinhardtii. The mutant is able to grow photoautotrophically but with a slower rate than the wild type strain. The heme group is covalently attached to the polypeptide, and the visible absorption spectrum of the mutant protein is identical to that of the native protein. The kinetics of electron transfer in the mutant were measured by flash kinetic spectroscopy. Our results show that the rate for the oxidation of cytochrome f was unchanged (t(12) = approximately 100 micros), but the half-time for the reduction of cytochrome f is increased (t(12) = 32 ms; for wild type, t(12) = 2.1 ms). Cytochrome b(6) reduction was slower than that of the wild type by a factor of approximately 2 (t(12) = 8.6 ms; for wild type, t(12) = 4.7 ms); the slow phase of the electrochromic band shift also displayed a slower kinetics (t(12) = 5.5 ms; for wild type, t(12) = 2.7 ms). The stability of the cytochrome b(6)f complex in the mutant was examined by following the kinetics of the degradation of the individual subunits after inhibiting protein synthesis in the chloroplast. The results indicate that the cytochrome b(6)f complex in the small domain deletion mutant is less stable than in the wild type. We conclude that the small domain is not essential for the biogenesis of cytochrome f and the cytochrome b(6)f complex. However, it does have a role in electron transfer through the cytochrome b(6)f complex and contributes to the stability of the complex.     

Reaction of ascorbic acid with cytochrome b561. Concerted electron and proton transfer.

Rate constants for reduction of cytochrome b561 by internal ascorbate (k0A) and oxidation by external ferricyanide (k1F) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b561 to ferricyanide (k1F) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (k0A), like the previously measured rate constant for reduction by external ascorbate (k-1A), is not very pH-dependent and is not consistent with reduction of cytochrome b561 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b561 is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome b561 by ferricyanide (k1F) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome b561 and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.     

Parallel pathways in cytochrome c(551) folding

The folding of cytochrome c(551) from Pseudomonas aeruginosa was previously thought to follow a simple sequential mechanism, consistent with the lack of histidine residues, other than the native His16 heme ligand, that can give rise to mis-coordinated species. However, further kinetic analysis reveals complexities indicative of a folding mechanism involving parallel pathways. Double-jump interrupted refolding experiments at low pH indicate that approximately 50% of the unfolded cytochrome c(551) population can reach the native state via a fast (10 ms) folding track, while the rest follows a slower folding path with populated intermediates. Stopped-flow experiments using absorbance at 695 nm to monitor refolding confirm the presence of a rapidly folding species containing the native methionine-iron bond while measurements on carboxymethylated cytochrome c(551) (which lacks the Met-Fe coordination bond) indicate that methionine ligation occurs late during folding along the fast folding track, which appears to be dominant at physiological pH. Continuous-flow measurements of tryptophan-heme energy transfer, using a capillary mixer with a dead time of about 60 micros, show evidence for a rapid chain collapse within 100 micros preceding the rate-limiting folding phase on the milliseconds time scale. A third process with a time constant in the 10-50 ms time range is consistent with a minor population of molecules folding along a parallel channel, as confirmed by quantitative kinetic modeling. These findings indicate the presence of two or more slowly inter-converting ensembles of denatured states that give rise to pH-dependent partitioning among fast and slow-folding pathways.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas sphaeroides

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.     

Spectroelectrochemical study of cytochrome c oxidase: pH and temperature dependences of the cytochrome potentials. Characterization of site-site interactions

The cytochrome a and a3 sites in uninhibited, detergent-solubilized cytochrome c oxidase have been studied under a wide range of conditions using thin-layer spectroelectrochemistry. The observed absorbance changes at the alpha and Soret absorbance maxima have been used together to estimate the extents of reduction of cytochromes a and a3, using the absorbance properties of these cytochromes deduced from previous measurements employing ligand inhibition of cytochrome a3. The resulting Nernst plots, combined with the results of parallel studies on the carbon monoxide-inhibited enzyme (Ellis, W. R., Jr., Wang, H., Blair, D. F., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 161-167; Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 167-171), indicate that the cytochrome a site participates in anticooperative thermodynamic interactions which involve all three of the other metal sites in the protein. Using an analysis which resolves the intrinsic thermodynamic properties of the cytochromes from the effects of the intersite interactions, the pH, temperature, and ionic strength dependences of the cytochrome reduction potentials have been measured. The standard entropy of reduction of cytochrome a in the native enzyme is large and negative, in agreement with measurements on the carbon monoxide-inhibited enzyme. The reduction potential of cytochrome a is only moderately (less than -30 mV/pH unit) dependent upon pH, which implies that its reduction is linked to the uptake, on the average, of only about 0.5 protons at pH 7.0, and significantly less at the higher pH values relevant to the mitochondrial matrix. The thermodynamic properties of cytochrome a3 were found to be different in the two enzyme batches studied: in one batch, the cytochrome a3 reduction potential decreased steeply (about -56 mV/pH unit) with increasing pH, indicating stoichiometric (1 H+/e-) coupling of protonation to reduction. In the other batch, the cytochrome a3 potential was insensitive to pH below pH 7.5 and decreased at higher pH values in a manner suggesting coupling to an ionizable group with pKa near 7.8. The temperature dependence of the cytochrome a3 reduction potential indicates that its standard entropy of reduction is more positive than that of myoglobin, another high-spin metalloprotein heme, and significantly more positive than that of cytochrome a.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of the superoxide-producing oxidase of neutrophils. O2 is necessary for the fast reduction of cytochrome b-245 by NADPH.

A soluble oxidase from phorbol-stimulated pig neutrophils contained FAD and cytochrome b-245. A typical preparation produced 13.03 mol of superoxide (O2-.) X S-1 X mol of cytochrome b-1 (348 nmol X min-1 X mg of protein-1). In the aerobic steady state, cytochrome b was 8.9% reduced. Steady-state cytochrome b reduction was absent from extracts of unstimulated cells; Km values for NADPH, for O2-. production and cytochrome b reduction were similar. The calculated aerobic rate of cytochrome b reduction was equal to the measured rate of O2-. production in a variety of preparations and in the presence of a range of inhibitors. Under anaerobic conditions the rate was slow: O2 is apparently required for rapid electron flow into the oxidase complex. Cytochrome b is shown to be kinetically competent to act as part of the O2-.-generating complex.     

A comparative laser-flash absorption spectroscopy study of algal plastocyanin and cytochrome c552 photooxidation by photosystem I particles from spinach.

Laser-flash kinetic absorption spectroscopy has been used to compare the rate constants for electron transfer from reduced plastocyanin and cytochrome c552, obtained from the green alga Monoraphidium braunii, to photooxidized P700 (P700+) in photosystem I (PSI) particles from spinach Sigmoidal protein concentration dependence for the observed electron-transfer rate constants are obtained for both proteins. In the absence of added salts, the P700+ reduction rate increases as the pH decreases from approximately 8 to 5.5, then decreases to pH 3.5, this effect being more pronounced with cytochrome c552 than with plastocyanin. At neutral pH, plastocyanin is a more efficient electron donor to P700+ than cytochrome c552, whereas at pH 5.5, which is closer to physiological conditions, the two redox proteins react with approximately equal rate constants. In the presence of increasing concentrations of added salts, the P700+ reduction rate constants for both proteins increase at pH greater than 5.5, but decrease at pH less than 4. At neutral pH, the observed rate constants for both algal proteins have a biphasic dependence on sodium chloride concentration, increasing in a parallel manner with increasing salt concentration, reaching a maximum value at 50 mM NaCl, then decreasing. A similar biphasic dependence is obtained with magnesium chloride, but in this case the maximum value is reached at salt concentrations ten times smaller, suggesting a specific role for the divalent cations in the electron-transfer reaction.     

Rate of electron transfer between cytochrome b561 and extravesicular ascorbic acid

Cytochrome b561 transfers electrons across secretory vesicle membranes in order to regenerate intravesicular ascorbic acid. To show that cytosolic ascorbic acid is kinetically competent to function as the external electron donor for this process, electron transfer rates between cytochrome b561 in adrenal medullary chromaffin vesicle membranes and external ascorbate/semidehydroascorbate were measured. The reduction of cytochrome b561 by external ascorbate may be measured by a stopped-flow method. The rate constant is 450 (+/- 190) M-1 s-1 at pH 7.0 and increases slightly with pH. The rate of oxidation of cytochrome b561 by external semidehydroascorbate may be deduced from rates of steady-state electron flow. The rate constant is 1.2 (+/- 0.5) x 10(6) M-1 s-1 at pH 7.0 and decreases strongly with pH. The ratio of the rate constants is consistent with the relative midpoint reduction potentials of cytochrome b561 and ascorbate/semidehydroascorbate. These results suggest that cytosolic ascorbate will reduce cytochrome b561 rapidly enough to keep the cytochrome in a mostly reduced state and maintain the necessary electron flux into vesicles. This supports the concept that cytochrome b561 shuttles electrons from cytosolic ascorbate to intravesicular semidehydroascorbate, thereby ensuring a constant source of reducing equivalents for intravesicular monooxygenases.     

Inhibition of electron transfer through the cytochrome b-c 1 complex by nitric oxide in a photodenitrifier,Rhodopseudomonas sphaeroides forma sp. denitrificans

The effects of nitric oxide (NO) on electron transfer were studied with a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans. NO inhibited the oxidation of cytochrome c induced by continuous illumination in intact cells. NO inhibited the re-reduction of cytochrome c, the slow phase of the carotenoid bandshift, and the oxidation of cytochrome b after a flash illumination, suggesting that NO inhibited the photosynthetic cyclic electron transfer through the cytochrome b-c ₁ region. NO also inhibited the nitrite (NO ₂ ^- ) and NO reductions with succinate as the electron donor in intact cells, but did not inhibit the NO ₂ ^- and NO reductions in chromatophore membranes with ascorbate and phenazine methosulfate as the electron donors. NO reversibly inhibited the ubiquinol: cytochrome c oxidoreductase of the membranes, suggesting that NO inhibited the electron transfer through the cytochrome b-c ₁ region and that the cytochrome b-c ₁ complex also was involved in the electron transport in both NO ₂ ^- and NO reductions. The catalytic site of NO reduction was distinct from the inhibitory site of NO.Abbreviations UHDBT 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole - UHNQ 3-undecyl-2-hydroxy-1,4-naphthoquinone - MOPS 3-(N-morpholino)propane-sulfonic acid - PMS phenazine methosulfate - DCIP 2,6-dichlorophenol indophenol - DDC diethyl-dithiocarbamate     

Photoreactivation of the cytochrome oxidase complex with cyanide: the reaction of heme a3 photoreduction.

Electron transfer activity of isolated cytochrome oxidase inhibited by low concentrations of cyanide by 93-95% was shown to rise no less than three times under exposure to visible light. Irradiation with visible light was found to increase the rate of reduction of cytochrome oxidase heme groups in the presence of sodium dithionite. Based on these results, it is suggested that the modification of the catalytic and spectral characteristic of the cytochrome oxidase-cyanide complex is due to the photostimulation of the intramolecular electron transport at the interheme (heme a heme a3) transfer stage, i.e., is caused by photoreduction of the enzyme's heme a3-CN complex.     

Interaction between cytochrome c and cytochrome c peroxidase: excited-state reactions of zinc- and tin-substituted derivatives

The effect of cytochrome c peroxidase (CCP) and apoCCP on the fluorescence and phosphorescence of Zn and Sn cytochrome c (cyt c) and the effect of cyt c on the fluorescence and phosphorescence of Zn CCP were examined. We found the following: The fluorescence yields of Zn and Sn cyt c were quenched by about 20% by CCP, consistent with energy transfer between the two chromophores with a separation of about 1.8 nm. The phosphorescence spectrum of Zn cyt c (but not Sn cyt c) shifts by 20 nm to the blue upon complexation with either CCP or apoCCP; at the same time the phosphorescence lifetime of Zn cyt c decreases from 12 +/- 2 to 6 ms with apoCCP addition. Zn CCP phosphorescence decay increases from 8.3 to 9.1 ms upon addition of poly(L-lysine) used to mimic cyt c. It is concluded from these results that binding of the redox partner or an analogue to Zn CCP and Zn cyt c results in a conformational change. The respective phosphorescence lifetimes of Zn and Sn cyt c were 13 and 3 ms in the absence of CCP and 1.6 and 1.1 ms in the presence of CCP; this corresponds to a quenching rate due to CCP of 519 and 570 s-1, for Zn and Sn cyt c, respectively. The phosphorescence of Zn CCP is also affected by native cyt c but is dramatically less than the complementary pair; the quenching rate constant is 17 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the reduction and oxidation of cytochromes b6 and ƒ in isolated chloroplasts

Absorbance changes induced by flash illumination of a chloroplast suspension were monitored kinetically at selected wavelengths between 510 and 575 nm. Digital subtraction of the P518 component from the total transient signal permitted isolation of the responses due to cytochromes ƒ and b₆. The half rise time for cytochrome b₆ reduction (1.3±0.1 ms) was much greater than a previously reported value (< 10 μs); the half time for cytochrome b₆ oxidation was 35±4 ms. Cytochrome ƒ was oxidized with a half time of about 0.22 ms and the subsequent reduction occurred in two phases with half times of about 7.3 and 83 ms. These kinetic data show that cytochrome b₆ cannot be a primary electron acceptor in Photosystem 1. The rate of oxidation of cytochrome b₆ is consonant with this cytochrome being the source of electrons for the slower phase of cytochrome ƒ reduction.     

Control of proteoliposomal cytochrome c oxidase: the partial reactions

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith-Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by delta pH and by delta psi can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.     

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. II. Direct electron transfer between the reaction center and the bc1 complex and role of cytochrome c2

1. The cyclic photosynthetic chain of Rhodobacter capsulatus has been reconstituted incorporating into phospholipid liposomes containing ubiquinone-10 two multiprotein complexes: the reaction center and the ubiquinol-cytochrome-c2 reductase (or bc1 complex). 2. In the presence of cytochrome c2 added externally, at concentrations in the range 10-10(4) nM, a flash-induced cyclic electron transfer can be observed. In the presence of antimycin, an inhibitor of the quinone-reducing site of the bc1 complex, the reduction of cytochrome b561 is a consequence of the donation of electrons to the photo-oxidized reaction center. At low ionic strength (10 mM KCl) and at concentrations of cytochrome c2 lower than 1 microM, the rate of this reaction is limited by the concentration of cytochrome c2. At higher concentrations the reduction rate of cytochrome b561 is controlled by the concentration of quinol in the membrane, and, therefore, is increased when the ubiquinone pool is progressively reduced. At saturating concentrations of cytochrome c2 and optimal redox poise, the half-time for cytochrome b561 reduction is about 3 ms. 3. At high ionic stength (200 mM KCl), tenfold higher concentrations of cytochrome c2 are required for promoting equivalent rates of cytochrome-b561 reduction. If the absolute values of these rates are compared with those of the cytochrome-c2-reaction-center electron transfer, it can be concluded that the reaction of oxidized cytochrome c2 with the bc1 complex is rate-limiting and involves electrstatic interactions. 4. A significant rate of intercomplex electron transfer can be observed also in the absence of cytochrome c2; in this case the electron donor to the recation center is the cytochrome c1 of the oxidoreductase complex. The oxidation of cytochrome c1 triggers a normal electron transfer within the bc1 complex. The intercomplex reaction follows second-order kinetics and is slowed at high ionic strength, suggesting a collisional interaction facilitated by electrostatic attraction. From the second-order rate constant of this process, a minimal bidimensional diffusion coefficient for the complexes in the membrane equal to 3 X 10(-11) cm2 s-1 can be evaluated.