Electron transfer rate, flavocytochrome

Capeillere-Blandin, C., Barber, M. J., Bray, R. C. Differences in electron transfer rates among prosthetic groups between two homologous flavocytochrome b2 (L-lactate cytochrome c oxidoreductase) from different yeasts. In Flavins and flavoproteins (Massey, V., Williams, C. H. eds.) pp. 838-843, New York, Amsterdam, Oxford, Elsevier/North Holland 1982... 

The physiological pathway of electron transfer in flavocytochrome is from bound lactate to FMN, then FMN to 52-heme, and finally 52-heme to cytochrome c (Fig. 9) (2,11, 80,102). The first step, oxidation of L-lactate to pyruvate with concomitant electron transfer to FMN, is the slowest step in the enzyme turnover (103). With the enzyme from S. cerevisiae, a steady-state kinetic isotope effect (with ferricyanide as electron acceptor) of around 5 was obtained for the oxidation of dl-lactate deuterated at the C position, consistent with the major ratedetermining step being cleavage of the C -H bond (103). Flavocytochrome 52 reduction by [2- H]lactate measured by stopped-flow spectrophotometry resulted in isotope effects of 8 and 6 for flavin and heme reduction, respectively, indicating that C -H bond cleavage is not totally rate limiting (104). 

Rates of Electron Transfer for Flavocytochromes 62 om S. cerevisiae and H. anomala ... 

FIGURE 6. Schematic representation of the catalytic reaction cycle in flavocytochrome b2. Five redox intermediates of FCB2 during the oxidation of one molecule of lactate at a steady-state turnover rate of 100 sec and the reduction of two molecules of cytochrome c at the rate of 200sec° are shown. Step 4 is the rate limiting step in the steady state and the maximal rates of some of the other electron transfer steps are indicated. Reproduced from Daff et al., 1996 with permission. 

Fig. 13. Proposed mechanism of L-lactate oxidation by flavocytochrome 62- The rate-determining step of the reaction is k lk-i. Electron transfer from substrate carbanion to FMN is postulated as proceeding via a covalent intermediate alternatives to this are shown in Fig. 12. E, Enzyme S, substrate.

For H. anomala flavocytochrome 62 there is a detectable lag phase between flavin and heme reduction, indicating that intramolecular electron transfer from FMN to heme is partly rate limiting (66). Values for rate constants (at 5°C) for phase 1 and phase 2 in both H. anomala and S. cerevisiae enzymes (66) are listed in Table VI. Using temperature-jump methods, Tegoni et al. (125) studied the electron transfer from flavosemiquinone to heme and determined a rate constant (at 16°C) of 160 sec. This value was thought to correspond to the value of kcat, which is 225 see (at 16°C) (125). Tegoni et al. (125)... 

Another Class II P-450 redox system that has been extensively studied is the cytosolic flavocytochrome P-450 BM3 from Bacillus megaterium. BM3 is the fusion of a soluble P-450 domain with CPR." " " The FAD of BM3 is reduced by NADPH the electrons are transferred to FMN and then finally to the substrate-bound P-450 domain." " BM3 is the fastest reported P-450 monooxygenase,with the rate of hydride transfer from NADPH to FAD and the rate of electron transfer from FMN to heme several-fold above the mammalian P-450 redox systems." The structure of the full-length protein has yet to be solved, but the structure of the FAD- and NADPH-binding domain has been determined." This domain closely resembles rat liver CPR and contains several conserved residues implicated in NADPH binding and flavin reduction. 

Figures 1.28 and 1.29 display respectively the variation of the second-order rate constant with ionic strength for flavocytochrome b2 cyt.c and cytochrome b2 core cyt.c electron transfer, illustrated by the classical Debye-Hiickel plot defined by log k+ versus the square root of ionic strength ...

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