Some One Electron Reduction Products of Flavin Analogs: Cyanoisoalloxazines and Deazaisoalloxazines

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

Section: Methodsmentioning

confidence: 99%

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

Ahmad¹,

Cusanovich²,

Tollin³

1982

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“…Samples containing 4 X 10~5M 10-methylisoalloxazine and appropriate concentrations of the substrate (phenol or both phenol and quinone) dissolved in the solvent were placed in a 1 cm path-length cuvette, deoxygenated by bubbling with nitrogen gas for 15-20 min, and flash photolyzed. The flash photolysis experiments were conducted with a pulsed nitrogen laser pumped dye laser as previously described (Tollin et al, 1979). All samples were prepared in minimal light.…”

Section: Methodsmentioning

confidence: 99%

“…We have utilized laser flash photolysis techniques (Tollin et al, 1979) to investigate the solvent dependence of the following reactions (Scheme I). (a) The first reaction is electron transfer to the flavin triplet state (3F) from oxidized flavin (F) (self-quenching) and from 2,5-dimethylphenol (external quenching).…”

mentioning

confidence: 99%

Solvent effects on flavin electron transfer reactions

Ahmad¹,

Tollin²

1981

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The effects of solvent environment on the rates of several flavin redox reactions have been studied by using laser flash photolysis. These include electron transfer to the flavin triplet state (a measure of oxidized flavin electrophilicity) and the oxidation of flavin semiquinone by oxidized flavin radical, oxidized phenol radical, and quinone. The rate constant for triplet quenching by 2,6-dimethylphenol was found to be proportional to the inverse of solvent viscosity, as would be expected for a diffusional process. The flavin semiquinone yield due to flavin reduction during the quenching reaction was linearly dependent on the solvent dielectric constant. This implies the existence of a polar or charged intermediate along the reaction pathway. A similar effect of solvent dielectric was found for the self-quenching reaction, which produces semiquinone and an oxidized flavin radical. The rate constants for all three of the semiquinone oxidation reactions studied were found to exhibit a biphasic dependence on solvent dielectric, being virtually independent of dielectric at low values and sharply increasing at high values. This is interpreted in terms of a change in mechanism with solvent polarity. Specifically, we propose a neutral transition state and hydrogen atom transfer in low dielectric media and a dipolar transition state and electron transfer in high dielectric media. No specific effects of hydrogen-bonding interaction between flavin and solvent were observed for any of the processes studied. The mechanistic implications of these results for flavoenzyme catalysts are discussed.

“…When this was carried out in the presence of the oxidized or semiquinone forms of flavodoxin, the reduction of the latter species by the deazaflavin radical could be monitored. The photolysis apparatus has been previously described (Tollin et al, 1979). The exciting source was a N2-laser pumped dye [2-(lnaphthyl)-5-phenyloxazole] which produced a 10-ns pulse of light at 393 nm.…”

mentioning

confidence: 99%

Transient kinetics of electron-transfer reactions of flavodoxins

Jung¹,

Tollin²

1981

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Stopped-flow and laser photolysis methods have been used to investigate the rates of electron-transfer reactions of fully reduced riboflavin and the three oxidation states of Clostridium pasteurianum flavodoxin. Both normal and 7,8-dichloroflavin analogues were studied. Redox reagents included oxygen, ferricyanide, ferric EDTA, and several c-type cytochromes as oxidants and the semiquinone of 5-deazariboflavin as a reductant. The dependence of the rate of oxidation of the semiquinone form of the dichloro analogue flavodoxin upon oxidant concentration has provided clear evidence for the existence of a complex in the reaction pathway. Rate constant comparisons demonstrate that dichloro substitution decreases the rate of flavodoxin semiquinone oxidation by at least 1-2 orders of magnitude. The limiting first-order rate constants were found to be dependent on the redox potential of the oxidant, as would be predicted by theory if these were reflecting the actual electron-transfer reaction. Rate constant decreases upon chlorine substitution were also observed for the reduction of both oxidized and semiquinone forms of flavodoxin by deazariboflavin semiquinone. These results, considered in conjunction with the redox potential shift of the flavodoxin produced by the chlorine substitution, provide support for the hypothesis that electron transfer to and from the semiquinone form of the flavodoxin involves direct participation of the dimethylbenzene ring of the flavin. A comparison of oxidation rate constants for free and protein-bound fully reduced flavin suggests that the protein environment does not markedly influence coenzyme reactivity in this oxidation state.