Pyruvate reduction, flavocytochrome

Flavocytochrome b2 catalyses the oxidation of lactate to pyruvate at the expense of cytochrome C. After reduction of flavin (FMN) by the substrate, reducing equivalents are transferred to heme b2 and from there to cytochrome C602. The mechanism of this process has been studied603 at 5.0 °C by determining the D KIE in the FMN reduction using L-[2-2H]lactate and wild-type enzyme and also with the Y143F mutant prepared from transformed Escherichia coli604. Tritium IE in the conversion of [2-3-H]lactate to... 

The first step in the catalytic cycle of flavocytochrome i>2 is the oxidation of L-lactate to pyruvate and the reduction of the flavin. Our understanding of how this occurs has been dominated by what can only be described as the dogma of the carbanion mechanism. Although this mechanism for flavoprotein catalysed substrate oxidations is accepted by many, doubts remain, and the alternative hydride transfer process cannot be ruled out. The carbanion mechanism has been extensively surveyed in the past, reviews by Lederer (1997 and 1991) and Ghisla and Massey (1989) are recommended, and for this reason there is little point in covering the same ground in the present article in any great detail. 

EPR signals for both the flavosemiquinone radical and the low-spin ferric heme have been reported (65, 78-82). The flavosemiquinone signal, which is easily observed at 123 K, shows a typical g value of 2.0039 0.002 (65). The bandwidth, which is around 15 G, is very like that of an anionic, or red, semiquinone (65). The EPR signal of the low-spin ferric heme can be observed at low temperatures ( 28 K) and shows g values of 2.99, 2.22, and 1,47 (65), which are similar to those found for cytochrome 65 (81). EPR rapid freezing studies have allowed the amounts of semiquinone and ferric heme to be monitored during reduction of the enzyme by L-lactate (66). This has proved to be extremely useful in the development of kinetic schemes to describe the flow of electrons in the enzyme. The distance between the prosthetic groups in H. anomala flavocytochrome 62 has been estimated from EPR experiments and spin-lattice relaxation measurements (82). Pyruvate was used to stablize the flavosemiquinone and the effect on the signal of this species from oxidized and reduced heme was measured. The results indicated a minimum intercenter distance of 18-20 A (82). 

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). 

By considering the above-mentioned solution studies and the refined three-dimensional structure of the S. cerevisiae flavocytochrome 62 active site, Lederer and Mathews proposed a scheme for the reverse reaction (the reduction of pyruvate) (39). They did not discuss how the transfer of electrons took place except to say that the structure did not rule out the possibility of a covalent intermediate (39). Ghisla and Massey (116) considered the anionic flavin N5 to be too close to the pyruvate carbonyl (3.7 A) without the formation of a covalent adduct taking place. Covalent intermediates between substrate and flavin have been observed for lactate oxidase (117, 118) and o-amino acid... 

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