Ferricyanide, flavocytochrome

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). 

As mentioned in Section V,A, heme-deficient (dehemo) flavocytochrome 2 has no cytochrome c reductase activity (126), but retains some ferricyanide reductase activity. These results are consistent with 2-heme being essential for cytochrome c reduction and also indicate that ferricyanide and cytochrome c are reduced by different mechanisms. 

Cytochrome c has often been used as an external electron acceptor in steady-state experiments with flavocytochrome 62 and usually gives rise to lower specific activities than would be found if ferricyanide was used as acceptor. The differences between cytochrome c and ferricyanide as electron acceptors are discussed further in the next section. Values of Km for cytochrome c from steady-state measurements are very dependent on the nature of the buffer used, with values ranging from 10 fj-M (10 mM Tris-HCl, pH 7.5,1 = 0.10 M NaCl) 134) to 180 fj.M (100 mM phosphate, pH 7.2) 135) seen with intact flavocytochrome 62 from S. cerevisiae. It is perhaps not surprising that the Km for cytochrome c is markedly affected in phosphate buffer considering the propensity for phosphate to bind to cytochrome c 136). 

Ferricyanide is the most commonly used electron acceptor in steady-state kinetic experiments on flavocytochrome 62. How is ferricyanide reduced by the enzyme Ogura and Nakamura suggested that ferricyanide could accept electrons only from the 62 heme (79). This is clearly incorrect, because dehemoflavocytochrome 62 and the isolated flavode-hydrogenase domain can still function as ferricyanide reductases, though at somewhat lower efficiency 51, 126). These results imply that ferricyanide can accept electrons from both flavohydroquinone and flavosemiquinone as well as heme. In heme-free cleaved enzyme from S. cerevisiae it was calculated that ferricyanide was reduced around 20 times faster by flavosemiquinone than by flavohydroquinone 126). This would mean that in the holoenzyme, reduction of ferricyanide would occur rapidly from heme and flavosemiquinone. The fact that ferricyanide is reduced by both 62 heme and flavosemiquinone, and that cytochrome c is reduced only by 62 heme, might be an explanation for the observation that specific activities of the enzyme determined with cytochrome c are usually somewhat lower than those determined with ferricyanide. 

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