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).     

Heterogeneous pools of cytochrome c2 in photo-denitrifying cells of Rhodobacter sphaeroides forma sp. denitrificans

When cells of the denitrifying phototrophic bacterium Rhodobacter sphaeroides forma sp. denitrificans were grown anaerobically under illumination in the presence of nitrate, the content of photosynthetic reaction centers per cellular protein was less than that in cells grown photosynthetically without nitrate under the same light intensity. The contents of cytochromes c1 and c2, which work in both photosynthetic and denitrifying electron transport systems, were almost constant, being independent of the presence of nitrate during growth. Consequently, the ratio of cytochromes c1 and c2 to the reaction center was more than three in the photo-denitrifying cells, whereas it was close to one in the photosynthetic cells under light-limiting conditions. In spite of the excess of cytochromes c1 + c2 over the reaction center in the photo-denitrifying cells, all cytochromes c1 + c2 were oxidized by illumination within hundreds of milliseconds in the presence of antimycin. When glycerol was added to increase the viscosity in the periplasm, biphasic oxidation of cytochromes c1 + c2 was apparent in the photo-denitrifying cells with repetitive flashes. The fast phase oxidation, which took place instantaneously (less than 1 ms) after the first and second flashes, showed a similar pattern to the oxidation in the light-limiting photosynthetic cells. The rate of the slow phase oxidation was sensitive to viscosity and was thought to reflect a diffusion-controlled second-order reaction between cytochrome c2 and the reaction center. The biphasic oxidation of cytochromes c1 + c2 suggests that these cytochromes exist in the photo-denitrifying cells as two different pools in relation to the reaction center.     

Oxidation of cytochrome c2 and of cytochrome c by reaction centers of Rhodospirillum rubrum and Rhodobacter sphaeroides. The effect of ionic strength and of lysine modification on oxidation rates

The oxidation of cytochrome c2 by the photooxidized reaction center bacteriochlorophyll, P+-870, in chromatophores of Rhodospirillum rubrum can be described using second-order kinetics at all ionic strengths. In a system consisting of isolated R. rubrum reaction centers and purified R. rubrum cytochrome c2, the oxidation of cytochrome c2 also follows second-order kinetics. In both cases, the reaction rates at low ionic strength are weakly dependent on the ionic strength. The data suggest that the cytochrome remains mobile at very low ionic strength, since the observed kinetics can be easily explained assuming no significant tight binding of cytochrome c2 to the reaction center. In a system consisting of equine cytochrome c and reaction centers of either R. rubrum or Rhodobacter sphaeroides, the cytochrome c oxidation rate depends more strongly on the ionic strength. The high reaction rates at low ionic strength suggest that a significant portion of the cytochrome is bound. Using equine cytochrome c derivatives modified at specific lysine residues, it was shown that both R. rubrum and Rb. sphaeroides reaction centers react with equine cytochrome c through its exposed heme edge.     

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)     

Cytochrome c and c2 binding dynamics and electron transfer with photosynthetic reaction center protein and other integral membrane redox proteins

To further the understanding of the details of c-type cytochrome action as a redox carrier between major electron-transfer proteins, the single-turnover kinetics time course of cytochrome c and cytochrome c2 oxidation by flash-activated photosynthetic reaction center (purified from the bacterium Rhodobacter sphaeroides) has been examined under a wide variety of conditions of concentration, ionic strength, and viscosity with reaction center present in detergent dispersion and phosphatidylcholine proteoliposomes. We find that the three-state model proposed by Overfield and Wraight [Overfield, R. E., & Wraight, C. A. (1980) Biochemistry 19, 3322-3327] is generally sufficient to model the kinetics time course; many similarities are found with the cytochrome c-cytochrome c oxidase interaction in mitochondria. Further, we find the following: (1) Significant "product inhibition" by oxidized cytochrome c (c2) bound to the reaction center is apparent. (2) The viscosity sensitivity of the electron transfer into the reaction center from bound cytochrome c (c2) suggests a physical interpretation of the distal state. (3) The exchange dynamics of oxidized and reduced cytochrome c (c2) are similar regardless of the state of activation of the reaction center. (4) Preferential binding of the oxidized form of cytochrome c is revealed upon reconstitution of the reaction center into neutral lipid vesicles, permitting an independent confirmation of the binding suggested by the kinetics. (5) Flash-activated electron-transfer kinetics in reaction center hybrid protein systems have shown that diffusion and competitive binding characterize the behavior of cytochrome c as a redox carrier between the reaction center protein and either the cytochrome bc1 complex or the cytochrome c oxidase.     

Interaction between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membrane of phosphatidylcholine vesicles.

Cytochrome P-450 and NADPH-cytochrome P-450 REDUctase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphoaditylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80--90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the sysTEM IN A SIMILAR WAY TO THE FAST-PHASE REDUCTION OF CYTOCHROME P-450, though the latter is not the rate-limiting step of the overall reaction.     

Role of specific lysine residues in binding cytochrome c2 to the Rhodobacter sphaeroides reaction center in optimal orientation for rapid electron transfer

The role of specific lysine residues in facilitating electron transfer from Rhodobacter sphaeroides cytochrome c2 to the Rb. sphaeroides reaction center was studied by using six cytochrome c2 derivatives each labeled at a single lysine residue with a carboxydinitrophenyl group. The reaction of native cytochrome c2 at low ionic strength has a fast phase with a half-time of 0.6 microseconds that has been assigned to the reaction of bound cytochrome c2 [Overfield, R.E., Wraight, C.A., & DeVault, D. (1979) FEBS Lett. 105, 137]. Modification of lysine-55 did not affect the half-time of this phase but decreased the apparent binding constant by a factor of 2. The derivatives modified at lysines-10, -88, -95, -97, -99, -105, and -106 surrounding the heme crevice did not show any detectable fast phase but only slow second-order phases due to the reaction of solution cytochrome c2. These lysines thus appear to be involved in binding cytochrome c2 to the reaction center in an optimal orientation for electron transfer. The involvement of lysines-95 and -97 is especially significant, since they are located in an extra loop comprising residues 89-98 that is not present in eukaryotic cytochrome c. The reactions of horse cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or [(trifluoromethyl)phenyl]carbamoyl were also studied. The derivatives modified at lysines-22, -55, -88, and -99 far removed from the heme crevice had nearly the same half-times for the fast phase as native cytochrome c, 6 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the c-cytochromes in chromatophores from Rhodopseudomonas sphaeroides as a function of the concentration of cytochrome c2. Influence of this concentration on the oscillation of the secondary acceptor of the reaction centers QB

The oxidation kinetics of Cyt c1 and c2 have been measured in normal chromatophores and in chromatophores fused with liposomes in order to increase the internal volume. The kinetics of Cyt c1 oxidation were found to be dependent on Cyt c2 concentration. The initial rate of Cyt c1 oxidation decreased after fusion by a factor of about two, indicating a process dependent on diffusion. The results do not allow a clear distinction between a diffusion of Cyt c2 along the inner membrane surface or through the inner volume of the vesicle; two- and three-dimensional models are discussed. In contrast to Cyt c1, the kinetics of oxidation of Cyt c2 were not influenced by changes in concentration. It is concluded that reduced Cyt c2 is preferentially bound to the reaction centers. A binary pattern as a function of flash number from the dark-adapted state was measured in the turn-over of the two-electron gate of the reaction center. In chromatophores with more than 0.5 cytochrome c2 molecules per reaction center, this binary pattern titrated out with a midpoint around 340 mV on reduction of the suspension. In experiments with chromatophores with a low Cyt c2 content, or with spheroplast-derived vesicles which had lost Cyt c2, the binary oscillation in the two-electron gate could be observed at much lower potentials. The results suggest that the binding of reduced cytochrome c2 modifies the behavior of the two-electron gate. A model in which reaction center dimers are stabilized by Cyt c2 is proposed to explain the effect.     

Structural studies on reconstituted reaction center-phosphatidylcholine membranes.

Reaction center protein, isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26 mutant, was incorporated into phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. Cytochrome c, added to preformed reaction center-phosphatidylcholine vesicles, rapidly reduced up to 90% of the laser-generated (BChl)2+ of the reaction center (with kinetics of electron transfer similar to those in the chromatophore membrane) which suggests that the portion of the reaction center which accommodates functional cytochrome c binding sites is exposed predominantly on the exterior of the vesicles. Unit cell electron density profiles were derived from lamellar X-ray diffraction from oriented reaction center-phosphatidylcholine membrane multilayers at varying lipid/protein ratios. The analysis of these profiles showed that the reaction center protein incorporates into the phosphatidylcholine membrane with unique sidedness and that the profile of the reaction center protein itself is asymmetric and spans the membrane.     

Interaction between NADPH-cytochrome p-450 reductase and cytochrome p-450 in the membrane of phosphatidylcholine vesicles

Cytochrome P-450 and NADPH-cytochrome P-450 reductase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphatidylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80–90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the system in a similar way to the fast-phase reduction of cytochrome P-450, though the latter is not the rate-limiting step of the overall reaction.     

The oxidation of ferrocytochrome c in nonbinding buffer.

The apparent equilibrium constant and rate of oxidation was investigated for the reaction of cytochrome c with iron hexacyanide. It was found that if horse heart ferricytochrome c was exposed to ferricyanide (to oxidize traces of reduced protein) the cytochrome subsequently, even after extensive dialysis, had an apparent equilibrium constant different from that of electrodialyzed protein. The effect of ferricyanide ion apparently cannot be removed by ordinary dialysis. The ionic strength dependence of the apparent equilibrium constant and bimolecular oxidation rate constant was measured in the range 1--200 mM using Tris--cacodylate or potassium phosphate buffers at pH 7.0, and electrodialyzed horse heart cytochrome c. The oxidation reaction proceeded very rapidly. Extrapolated to zero ionic strength, kox (approximately 9 X 10(9) M-1 S-1) was about 7% of that calculated for a diffusion-limited reaction. Since the exposed heme edge occupies only the order of 3% of the surface area, electron transfer apparently results at nearly every collision with the active-site region. An effective charge of + 7.8 units was estimated for the oxidation reaction. The rate of oxidation of Pseudomonas aeruginosa c551 was much slower (kox at mu = 0 was the order of 6 X 10(3)), and was not consistent with diffusion-limited kinetics.     

Studies on partially reduced mammalian cytochrome oxidase reactions with ferrocytochrome c.

The kinetics of the electron-transfer process which occurs between ferrocytochrome c and partially reduced mammalian cytochrome oxidase were studied by the rapid spectrophotometric techniques of stopped flow and temperature jump. Stopped-flow experiments showed initial very fast extinction changes at 605 nm and at 563 nm, indicating the simultaneous reduction of cytochrome a and oxidation of ferrocytochrome c. During this 'burst' phase, say the first 50 ms after mixing, it was invariably found that more cytochrome c had been oxidized than cytochrome a had been reduced. This discrepancy in electron equivalents may be accounted for by the rapid reduction of another redox site in the enzyme, possibly that associated with the extinction changes observed at 830 nm. During the incubation period in which the partially reduced oxidase was prepared, the rate of reduction of cytochrome a by ferrocytochrome c, at constant reactant concentrations, decreased with time. Temperature-jump experiments showed the presence of two relaxation processes. The faster of the two phases was assigned to the electron-transfer reaction between cytochrome c and cytochrome a. A study of the concentration-dependence of the reciprocal relaxation time for this phase yielded a rate constant of 9 X 10(6)M-1-s-1 for the electron transfer from cytochrome c to cytochrome a, and a value of 8.5 X 10(6)M-1-s-1 for the reverse reaction. The equilibrium constant for the electron-transfer reaction is therefore close to unity. The slower phase has been interpreted as signalling the transfer of electrons between cytochrome a and another redox site within the oxidase molecule.     

Cytochrome C (Fe2+) as a competitive inhibitor of NADPH-dependent reduction of cytochrome P450 LM2: locating protein-protein interaction sites in microsomal electron carriers.

The kinetics of NADPH-dependent reduction of cytochrome P450 LM2 in the soluble monomeric reconstituted system in the absence of any substrate is shown to be monophasic. We show that ferrous cytochrome c acts as a competitive inhibitor of the reduction. In the presence of 1 mM benzphetamine an additional extremely fast phase was observed. Under these conditions ferrous cytochrome c was found to be a competitive inhibitor of the slow phase of the reduction process, which accounted for 80% of the total reduction amplitude. Inhibition experiments yield a dissociation constant for the LM2-reductase complex of 3.0 +/- 1.5 microM. This constant was the same both in the presence and in the absence of benzphetamine. Based on these data we conclude that cytochromes P450 and c bind to the same center on the NADPH-cytochrome P450 reductase molecule. Comparative analysis of the amino acid sequences reveals a detectable similarity between cytochrome c and cytochrome P450 LM2 at positions 68-87 and 121-145, respectively. In addition, a substantial similarity was shown for sequence fragments 204-224 of NADPH-cytochrome P450 reductase and 40-60 of cytochrome b5. Based on these findings a hypothesis for the location of the centers of intermolecular interactions on the molecules of cytochrome P450 LM2 and NADPH-cytochrome P450 reductase is proposed.     

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.     

Intrinsic uncoupling in proton-pumping cytochrome c oxidase: pH dependence of cytochrome c oxidation in coupled and uncoupled phospholipid vesicles

The pH dependence of the transient aerobic kinetics of cytochromes c and a has been investigated with cytochrome oxidase reconstituted in phospholipid vesicles in the absence and presence of an uncoupler and an ionophore. The cytochrome a reduction level immediately after the burst phase was 60-80% and was not significantly changed by the addition of uncoupler and/or ionophore. The coupled rate of ferro-cytochrome c oxidation increases linearly with decreasing pH in the range 8.4-5.4. The increase in rate on uncoupling becomes less with decreasing pH and low cytochrome c concentration, being almost zero at pH 5.4. The coupled rate is increased by a lowering of the outside pH when the inside pH is constant. Varying the inside pH with a constant outside pH of 7.4 has little effect on the rate. It is suggested that the electrochemical potential has two separate effects on the coupled rate: the pH gradient mainly slows down the intramolecular electron transfer, but the membrane potential also lowers the second-order rate constant for the reaction with cytochrome c. The results are interpreted in terms of a model in which protonation of an acid-base group with a pKa of 6.4 from the inside increases the catalytic constant. Protonation from the outside, on the other hand, leads to an intrinsic uncoupling, because the protonated enzyme in the output state can return to the input state. This has no adverse physiological effect, since it becomes significant only at pH values well below 7.     

Electrostatic effects on the kinetics of electron transfer reactions of cytochrome c caused by binding to negatively charged lipid bilayer vesicles.

The effect of binding reduced tuna mitochondrial cytochrome c to negatively charged lipid bilayer vesicles at low ionic strength on the kinetics of electron transfer to various oxidants was studied by stopped-flow spectrophotometry. Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenanthroline ion, whereas the rate of reaction with the negatively charged ferricyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants such as benzoquinone and Rhodospirillum rubrum cytochrome c2 were unaffected by binding to vesicles, suggesting little or no effect of membrane association on cytochrome structure or accessibility of the heme center. The kinetic effects were largest at lower cytochrome c to vesicle ratios, where there was a greater degree of exposure of negatively charged regions on the membrane. The reduction of cobalt phenanthroline and ferricyanide by bound cytochrome c proceeded by nonexponential kinetics, as compared with the monophasic kinetics observed in the absence of vesicles. This was probably due to the heterogeneous distribution of vesicle sizes which exists at a given lipid to protein ratio. Nonlinear oxidant concentration dependencies were observed for cobalt phenanthroline oxidation of membrane-bound cytochrome c, consistent with a (minimal) two-step kinetic mechanism involving association of the oxidant with the membrane followed by electron transfer. Based on a comparison of second-order rate constants as a function of lipid to protein mole ratio, binding of cytochrome c to the bilayer increased the efficiency of the cobalt phenanthroline reaction by a factor of approximately 500 at the highest lipid:protein ratio used. The results suggest a mechanism involving attractive and repulsive electrostatic interactions between the negatively charged bilayer and the electrically charged oxidants, which increase or decrease their effective concentrations at the membrane surface.     

Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide.

The increase in light emission of hydroperoxide-supplemented cytochrome c observed on addition of lipid vesicles was related to the degree of unsaturation of the fatty acids of the phospholipids: dipalmitoyl phosphatidylcholine was without effect, whereas dioleoyl phosphatidylcholine and soya-bean phosphatidylcholine enhanced chemiluminescence 2- and 3-fold respectively. Effects on light-emission were similar to those on O2 uptake. The chemiluminescence of the present system was sensitive to cyanide and to the radical trap 2,5-di-t-butylquinol, indicating a catlytic activity of cytochrome c and the presence of free-radical species respectively. Lipid-vesicle enhanced chemiluminescence showed different kinetic behaviours, apparently depending on unsaturation: three phases are described for soya-bean phosphatidylcholine, whereas only one phase was present in mixtures containing dipalmitoyl and dioleoyl phospholipids. Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide showed similar kinetic patterns with H2O2 and primary (ethyl) and tertiary (t-butyl and cumene) hydroperoxides. Participation of singlet molecular oxygen, mainly on the phase III of chemiluminescence, is suggested by the increase of light-emission by 1,4-diazabicyclo[2.2.2]-octane as well as by data from spectral analysis.     

Photooxidation of mitochondrial cytochrome c by isolated bacterial reaction centers: Evidence for tight-binding and diffusional pathways

The binding of horse heart mitochondrial cytochrome c to isolated reaction centers from Rhodopseudomonas sphaeroides is described. The kinetics of photooxidation of cytochrome c following a short actinic flash is compared to the expected binding state of the cytochrome at various concentrations and at different ionic strengths. At low ionic strength a very tight binding site (K_D10^-8 M) is apparent which is nonfunctional with respect to electron donation to the bound reaction center. This tightly bound cytochrome can react with another reaction center in a diffusion limited, second order process. A weaker binding site (K_D0.3 · 10^-6 M) is also boserved which is associated with rapid, first order electron transfer from cytochrome to reaction center. Both binding processes are weakened in the presence of salt and there is no detectable binding in 100 mM NaCl. Under such conditions cytochrome oxidation is entirely a diffusional, second order process. However, analysis of the flash intensity dependence of the extent of cytochrome oxidation, by the method of van Grondelle (van Grondelle, R. (1978) Ph.D. Thesis, State University, Leiden) indicated that the cytochrome was not freely mobile even in 100 mM NaCl, at least in the sense that reduced cytochrome only slowly dissociates from unactivated reaction centers. An overall kinetic/equilibrium scheme for cytochrome c binding and photooxidation by reaction centers is presented. This is very similar to that described earlier for cytochrome c₂ (Overfield, R.E., Wraight, C.A. and DeVault, D. (1979) FEBS Lett. 105, 137–142), but the tight binding site and associated diffusion controlled oxidation is unique to cytochrome c.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

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+.     

Electron transfer between primary and secondary donors in Rhodospirillum rubrum: evidence for a dimeric association of reaction centers

Light-induced oxidation of the primary electron donor P and of the secondary donor cytochrome c2 was studied in whole cells of Rhodospirillum rubrum in the presence of myxothiazole to slow down their reduction. 1. The primary and secondary electron donors are close to thermodynamic equilibrium during continuous illumination when the rate of the electron transfer is light-limited. This implies a long-range thermodynamic equilibration involving the diffusible cytochrome c2. A different behavior is observed with Rhodobacter sphaeroides R26 whole cells, in which the cytochrome c2 remains trapped within a supercomplex including reaction centers and the cytochrome b/c complex [Joliot, P., et al. (1989) Biochim. Biophys. Acta 975, 336-345]. 2. Under weak flash excitation, the reduction kinetics of the photooxidized primary donor are nearly exponential with a half-time in the hundred microseconds time range. 3. Under strong flash excitation, the reduction of the photooxidized primary donor follows a second-order kinetics. About half of the photooxidized primary donor is reduced in a few milliseconds while the remainder stays oxidized for hundreds of milliseconds despite an excess of secondary donors in their reduced form. The flash intensity dependence of the amplitude of the slow phase of P+ reduction is proportional to the square of the fraction of reaction centers that have undergone a charge separation.(ABSTRACT TRUNCATED AT 250 WORDS)