Pyruvate 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... 

Flavocytochrome b2 from Saccharomyces cerevisiae, a member of the FMN-dependent oxidoreductase superfamily, catalyzes the two-electron oxidation of lactate to pyruvate with subsequent electron-transfer to cytochrome c via the bound flavin [55], What distinguishes the enzyme from other family members is the N-terminal fusion of a heme-binding domain to the ySa-barrel structure, which hosts the primary active site. Rather than dumping the electrons from the reduced flavin hydroquinone onto molecular oxygen, they are transferred intramolecularly to the heme-binding domain and from there in a second intermolecular step to cytochrome c. 

As a respiratory enzyme, the production of flavocytochrome bi is induced by the presence of oxygen and, more specifically, L-lactate. In addition to providing pyruvate (the product of lactate oxidation) for the Krebs cycle, flavocytochrome bj also participates in a shorter respiratory chain that ultimately directs the energy gained from L-lactate dehydrogenation... 

FIGURE 1. The Physiological Role of S. cerevisiae Flavocytochrome 2- Flavocytochrome acts as a pyruvate source for hie Krebs cycle (A) and also transfers elech ons directly to cytochrome c (B). 

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. 

FIGURE 7. The Active Site in S. cerevisiae Flavocytochrome b - The dotted surfaces represent van der Waals radii. Flydrophobic contacts are shown between the methyl group of pyruvate and a number of alkyl amino acid side-chains. 

In 1915, Harden and Norris observed that dried yeast, when mixed with lactic acid, reduced methylene blue and formed pyruvic acid 4). Thirteen years later Bernheim prepared an extract from acetone-dried baker s yeast, which had lactate dehydrogenase activity (5). Bach and co-workers demonstrated that the lactate dehydrogenase activity was associated with a 6-type cytochrome, which they named cytochrome 62 (6). In 1954, the enzyme was crystallized, enabling the preparation of pure material and the identification of flavin mononucleotide as a second prosthetic group (2). Since then, significant advances have been made in the analysis of the structure and function of the enzyme. Much of the earlier work on flavocytochrome 62 has already been summarized in previous review articles (7-10). In this article we shall describe recent developments in the study of this enzyme, ranging fi om kinetic, spectroscopic, and structural data to the impact of recombinant DNA technology. 

The methyl group of pyruvate is in van der Waals contact with Leu 230 (not shown in Fig. 6), but is in an otherwise relatively uncrowded environment. This is borne out by the fact that flavocytochrome 62 can... 

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). 

As well as alternative substrates, there have been a number of studies on inhibitors of flavocytochrome 62- Known inhibitors include D-lactate (16, 92-95), pyruvate (16, 58, 60, 96), propionate (96), DL-man-delate (90, 91), sulfite (60), and oxalate (16, 60, 97). Values of K, for these inhibitors and the conditions and types of enzyme used can be found in the papers referenced above. All of the above inhibitors show typical competitive inhibition except pyruvate and oxalate, for which mixed inhibition has been observed (60, 97). Inhibition has also been reported for excess substrate with the intact enzymes from both S. cerevisiae (16) and H. anomala (92), though not apparently with the cleaved enzyme from S. cerevisiae (16). It is possible that inhibition by excess substrate arises either from different binding modes at the active site or from a second lower affinity binding site elsewhere on the enzyme. 

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). 

To understand the carbanion mechanism in flavocytochrome 62 it is useful to first consider work carried out on related flavoenzymes. An investigation into o-amino acid oxidase by Walsh et al. 107), revealed that pyruvate was produced as a by-product of the oxidation of )8-chloroalanine to chloropyruvate. This observation was interpreted as evidence for a mechanism in which the initial step was C -H abstraction to form a carbanion intermediate. This intermediate would then be oxidized to form chloropyruvate or would undergo halogen elimination to form an enamine with subsequent ketonization to yield pyruvate. The analogous reaction of lactate oxidase with jS-chlorolactate gave similar results 108) and it was proposed that these flavoenzymes worked by a common mechanism. Further evidence consistent with these proposals was obtained by inactivation studies of flavin oxidases with acetylenic substrates, wherein the carbanion intermediate can lead to an allenic carbanion, which can then form a stable covalent adduct with the flavin group 109). Finally, it was noted that preformed nitroalkane carbanions, such as ethane nitronate, acted as substrates of D-amino acid oxidase 110). Thus three lines of experimental evidence were consistent with a carbanion mechanism in flavoenzymes such as D-amino acid oxidase. 

To elucidate the mechanism in flavocytochrome 62, a series of reactions similar to those mentioned above were carried out. Urban et al. Ill) studied the reverse reaction in flavocytochrome 62 and demonstrated that dehydrohalogenation did indeed occur with bromo- and chloropyruvate, but not with fluoropyruvate. A partition ratio of 500 was found for oxidation versus elimination during the forward reaction (i.e., 1 mol of pyruvate formed for every 500 mol of halopyruvate) compared with a partition ratio of 2 for the reverse reaction 111). 

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... 

Flavocytochrome l>2 (Fbj) catalyzes the oxidation of L-lactate to pyruvate (Equation (6)). 

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