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G Tollin

Publications and source records attributed to G Tollin.

At least 109 records · Page 6Linked to original sources
PubMed1992

Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding.

Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Transport↗
PubMed1992

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.

Cytochrome c Group↗
PubMed1992

Laser flash photolysis studies of electron transfer mechanisms in cytochromes: an aromatic residue at position 82 is not required for cytochrome c reduction by flavin semiquinones or electron transfer from cytochrome c to cytochrome oxidase.

The influence of an aromatic side chain at position 82 of yeast iso-1-cytochrome c on the kinetics of its electron transfer reactions has been investigated using laser flash photolysis methods to compare a series of site-specific mutant cytochromes in their reduction by free flavin semiquinone and in electron transfer from reduced cytochrome to bovine cytochrome c oxidase. Although small (approximately 10%) but significant differences are observed between some of the mutants (S82, Y82, I82) and wild-type (F82) or G82 cytochrome in the second-order rate constant for reduction by lumiflavin semiquinone, these do not correlate with side-chain aromaticity. In the reaction between the ferrocytochromes and cytochrome c oxidase, significantly larger deviations from exponentiality are found for those mutants having aliphatic residues at position 82 than for wild type or Y82. We interpret the nonexponential behavior in terms of multiple orientations of the cytochromes within the oxidase binding site; the extent to which this occurs is apparently influenced by the character of the residue at position 82. However, a comparison of the average rate constants for electron transfer to cytochrome oxidase for the various mutants reveals that all are closely comparable to WT, except for I82 which is significantly slower (approximately threefold). These results, combined with those obtained previously from steady-state kinetic and thermodynamic measurements, suggest that the observed differences among the mutants are due to alterations in the mode of binding of the cytochrome to the oxidase, rather than to a specific requirement for the presence of an aromatic group at position 82.

Animals↗
PubMed1992

Electrostatic modulation of the kinetics of electron transfer from cytochrome c to cobalt phenanthroline by binding to lipid bilayers: effects of ionic strength and extent of incorporation of various negatively charged lipids.

The effect of electrostatically binding ferrous cytochrome c to anionic liposomes, composed of dimyristoyl phosphatidylglycerol (DMPG-), dioleoyl phosphatidyl-glycerol (DOPG-), or cardiolipin (CL2-) mixed with varying amounts of egg phosphatidylcholine (PC), on the kinetics of cytochrome oxidation by the positively charged cobalt phenanthroline ion has been measured using stopped-flow spectrophotometry. The rate of electron transfer is enhanced as much as 3000-fold by increasing the number of negatively charged binding sites on the liposome surface, and by as much as 1000-fold by decreasing the ionic strength of the buffer. The sigmoidal shape of the curve of rate constant vs mole percent anionic lipid is consistent with a positively cooperative effect of the negative surface charge. The rate stimulation is greater for DOPG(-)- and CL2(-)-containing liposomes than for DMPG- vesicles; this is most likely due to structural differences in the respective liposomes. The results do not provide any support for a role of structural changes in the bound cytochrome in influencing oxidation kinetics, a possibility suggested by recent spectroscopic measurements, although relatively small conformational effects cannot be completely ruled out.

Anions↗
PubMed1991

Unimolecular and bimolecular oxidoreduction reactions involving diprotein complexes of cytochrome c and plastocyanin. Dependence of electron-transfer reactivity on charge and orientation of the docked metalloproteins.

Cytochrome c and plastocyanin form an electrostatic complex, which can be reinforced by amide bonds in the presence of a carbodiimide. Besides this cross-linking, carbodiimide also converts carboxylate side chains into neutral N-acylurea groups. Four derivatives of the covalent diprotein complex, which differ in the degree of this charge neutralization, are separated by cation-exchange chromatography. Electron-transfer reactions at different ionic strengths involving the electrostatic complex and the four derivatives of the covalent complex are studied by laser flash photolysis with flavin semiquinones as reducing agents. The reactivity of the associated proteins toward external reductants cannot be predicted simply on the basis of this reactivity of the separate proteins. Qualitative analysis of the dependence on ionic strength of the reactions between FMN semiquinone and the covalent derivatives indicates sites at which this reductant interacts with the cross-linked proteins. The surprisingly small steric shielding of the protein redox sites in the covalent complex, as deduced from the reactions at high ionic strength, may indicate that the proteins have multiple reaction domains on their surfaces or that the complex is dynamical or both. The intracomplex (unimolecular) electron-transfer reaction is fast in the electrostatic complex (ket = 1300 +/- 200 s-1) but undetectably slow in each of the four derivatives of the covalent complex (ket less than 0.2 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome c Group↗
PubMed1991

Transient kinetics of flavin-photosensitized oxidation of reduced redox proteins. Comparison of c-type cytochromes and plastocyanins.

We have used laser flash photolysis to investigate the kinetics of oxidation of reduced plastocyanins obtained from spinach and the green alga Monoraphidium braunii by the triplet states of lumiflavin, riboflavin and FMN. We have compared the results of these experiments with the kinetics of reduction of the oxidized forms of these proteins by the corresponding flavin semiquinones, as well as with the kinetics of flavin oxidation and reduction of cytochrome c552 (Class I c-type cytochrome, generic name c553) from Monoraphidium. In all cases, the rate constants for oxidation were one or two orders of magnitude larger than for reduction, consistent with the greater thermodynamic driving force for the oxidation reaction. Similar steric and electrostatic effects were observed for both reactions with all proteins, suggesting that the same (or closely adjacent) sites were being utilized for electron removal and entry. The two algal proteins were quite similar to one another in their redox properties, consistent with their physiological role of being able to substitute for one another in photosynthetic electron transport. In contrast, the algal plastocyanin was more reactive than the spinach protein in both oxidation and reduction, suggesting differences in their steric properties at the site of electron transfer.

Chlorophyta↗
PubMed1991

Kinetic comparison of reduction and intramolecular electron transfer in milk xanthine oxidase and chicken liver xanthine dehydrogenase by laser flash photolysis.

A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.

Animals↗
PubMed1991

Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding.

The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rate constants (9 x 10(8) M-1 s-1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding identical second-order rate constants (1.1 x 10(7) M-1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvate binding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process.

Electron Transport↗
PubMed1991

Laser flash photolysis study of intermolecular and intramolecular electron transfer in trimethylamine dehydrogenase.

Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1991

Direct measurement of intramolecular electron transfer between type I and type III copper centers in the multi-copper enzyme ascorbate oxidase and its type II copper-depleted and cyanide-inhibited forms.

Transient kinetics of reduction of zucchini squash ascorbate oxidase (AO) by lumiflavin semiquinone have been studied by using laser flash photolysis. Second-order kinetics were obtained for reduction of the type I copper with a rate constant of 2.7 X 10(7) M-1 s-1, which is comparable to that obtained with other blue copper proteins such as plastocyanin. Following reduction, the type I copper was reoxidized in a protein concentration independent (i.e., intramolecular) reaction (kobs = 160 s-1). Comparison with literature values for limiting rate constants in transient single-turnover kinetic experiments suggests that intramolecular electron transfer probably is the rate-limiting step in enzyme catalysis. The extent of reoxidation of type I copper was approximately 55%, which is consistent with the approximately equal redox potentials of the type I and type III copper centers. Neither azide nor fluoride caused any significant changes in kinetics, although they are enzyme inhibitors and are thought to bind to the type II copper. In contrast, cyanide caused a concentration-dependent decrease in the extent of intramolecular electron transfer (with no change in rate constant), and decreased the rate constant for reduction of the type I copper by a factor of 2. The apparent dissociation constant for cyanide (0.2-0.4 mM) is similar to that reported for inhibition of enzyme activity. Removal of the type II copper from AO only marginally affected the kinetics of electron transfer to type I copper (k = 3.2 x 10(7) M-1 s-1) and slightly increased the extent but did not alter the rate constant of intramolecular electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbate Oxidase↗
PubMed1991

Kinetics of electron transfer from thioredoxin reductase to thioredoxin.

The reduction of Escherichia coli thioredoxin by thioredoxin reductase was studied by stopped-flow spectrophotometry. The reaction showed no dependence on thioredoxin concentration, indicating that complex formation was rapid and occurred during the dead time of the instrument. The kobs for the reaction of approximately 20 s-1 probably reflects the rate of electron transfer from thioredoxin reductase to thioredoxin and agrees with the kcat observed by steady-state kinetics. The reaction rate was unaffected by increasing the ionic strength, suggesting a lack of electrostatic stabilization in the interaction of the two proteins. A mutant thioredoxin in which a positively charged lysine in the active-site region was changed to a glutamic acid residue resulted in an electrostatic destabilization. Thioredoxin K36E was still a substrate for the reductase, but binding was impaired so that the rate could be measured by stopped-flow techniques as reflected by a dependence on protein concentration. Raising the ionic strength in this reaction served to shield the negative charge and increased the rate of binding to the reductase.

Electron Transport↗
PubMed1991

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)

Animals↗
PubMed1991

Ionic strength dependence of the kinetics of electron transfer from bovine mitochondrial cytochrome c to bovine cytochrome c oxidase.

The effect of ionic strength on the one-electron reduction of oxidized bovine cytochrome c oxidase by reduced bovine cytochrome c has been studied by using flavin semiquinone reductants generated in situ by laser flash photolysis. In the absence of cytochrome c, direct reduction of the heme a prosthetic group of the oxidase by the one-electron reductant 5-deazariboflavin semiquinone occurred slowly, despite a driving force of approximately +1 V. This is consistent with a sterically inaccessible heme a center. This reduction process was independent of ionic strength from 10 to 100 mM. Addition of cytochrome c resulted in a marked increase in the amount of reduced oxidase generated per laser flash. Reduction of the oxidase at the heme a site was monophasic, whereas oxidation of cytochrome c was multiphasic, the fastest phase corresponding in rate constant to the reduction of the heme a. During the fast kinetic phase, 2 equiv of cytochrome c was oxidized per heme a reduced. We presume that the second equivalent was used to reduce the Cua center, although this was not directly measured. The first-order rate-limiting process which controls electron transfer to the heme a showed a marked ionic strength effect, with a maximum rate constant occurring at mu = 110 mM (1470 s-1), whereas the rate constant obtained at mu = 10 mM was 630 s-1 and at mu = 510 mM was 45 s-1. There was no effect of "pulsing" the enzyme on this rate-limiting one-electron transfer process. These results suggest that there are structural differences in the complex(es) formed between mitochondrial cytochrome c and cytochrome c oxidase at very low and more physiologically relevant ionic strengths, which lead to differences in electron-transfer rate constants.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Laser flash photolysis studies of the kinetics of reduction of ferredoxins and ferredoxin-NADP+ reductases from Anabaena PCC 7119 and spinach: electrostatic effects on intracomplex electron transfer.

The influence of electrostatic forces on the formation of, and electron transfer within, transient complexes between redox proteins was examined by comparing ionic strength effects on the kinetics of the electron transfer reaction between reduced ferredoxins (Fd) and oxidized ferredoxin-NADP+ reductases (FNR) from Anabaena and from spinach, using laser flash photolysis techniques. With the Anabaena proteins, direct reduction by laser-generated flavin semiquinone of the FNR component was inhibited by complex formation at low ionic strength, whereas Fd reduction was not. The opposite results were obtained with the spinach system. These observations clearly indicate structural differences between the cyanobacterial and higher plant complexes. For the complex formed by the Anabaena proteins, the results indicate that electrostatic forces are not a major contributor to complex stability. However, the rate constant for intracomplex electron transfer had a biphasic dependence on ionic strength, suggesting that structural rearrangements within the transient complex facilitate electron transfer. In contrast to the Anabaena complex, electrostatic forces are important for the stabilization of the spinach Fd:FNR complex, and changes in ionic strength had little effect on the limiting rate constant for intracomplex electron transfer. This suggests that in this case the geometry of the initial collisional complex is optimal for reaction. These results provide a clear illustration of the differing roles that electrostatic interactions may play in controlling electron transfer between two redox proteins.

Cyanobacteria↗
PubMed1991

Chlorophyll photosensitized electron transfer reactions in lipid vesicles: enhancement in yield of vectorial electron transfer across the bilayer from reduced cytochrome c to oxidized ferredoxin by addition of valinomycin plus potassium ion.

Chlorophyll photosensitized electron transfer across a vesicle bilayer from reduced cytochrome c in the inner compartment to oxidized ferredoxin in the outer compartment, using propylene diquat as a mediator, has been investigated using both steady-state and laser flash photolysis methods. One of the factors limiting the quantum yield is the transmembrane potential, which is formed during sample preparation and is increased by the electron transfer process across the membrane bilayer. This limitation can be diminished by the incorporation of valinomycin into the bilayer in the presence of potassium ion. The overall quantum yield can be approximately doubled (up to a total of 22% based on the chlorophyll triplet which is quenched, and 2.8% based on the absorbed quanta) by valinomycin addition. Another quantum yield limitation arises from the accumulation of oxidized cytochrome c in the inner aqueous compartment, which is formed as a consequence of the transbilayer electron transport process and can quench triplet chlorophyll on the inner side of the vesicle. The chlorophyll cation radical generated in this way can participate in the electron exchange equilibrium between chlorophyll molecules located within the bilayer, and thus inhibit electron flow from inside to outside. This acts to limit the extent of cytochrome c oxidation to less than or equal to 50% of the original amount.

Chlorophyll↗
PubMed1990

Flavin-photosensitized oxidation of reduced c-type cytochromes. Reaction mechanism and comparison with photoreduction of oxidized cytochromes by flavin semiquinones.

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin-cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.

Cytochrome c Group↗