Flavin Reduction and Substrate Oxidation

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. 

The fundamentals on which the carbanion mechanism is founded are the early studies on the related enzj mes D-amino acid oxidase (Walsh et al., 1972 and 1971), lactate oxidase (Walsh et al., 1973) and flavocytochrome bj (Urban and Lederer, 1985 Pompon and Lederer, 1985). It is interesting that all of the key work, which established the carbanion mechanism, was done before any 3-dimensional structures were available on the respective enzymes. 

The core requirement for the carbanion mechanism to operate is that an active-site base must abstract the a-carbon hydrogen of the substrate, as a proton, forming a carbanion intermediate (Lederer, 1991). This would then require the equivalent of two electrons to be transferred to the flavin either with or without the formation of a covalent intermediate between the a-carbon and the flavin N-5 (Ghisla and Massey, 1989). With this in mind, it is intriguing to find that the crystal structure of D-amino acid oxidase reveals that there is no residue correctly located to act as the active-site base required for the carbanion mechanism (Mattevi et al., 1996 Mizu-tani et al., 1996). In fact, the crystallographic information available is far more consistent with this enzyme operating a hydride transfer mechanism (Mattevi et al., 1996). If this is correct then the earlier experiments on d-amino acid oxidase, which were claimed to be diagnostic of a carbanion mechanism, are ealled into question. It is important to note that similar experiments were used to provide support for a carbanion mechanism in the ease of flavocytochrome b2- 

The high-level expression of recombinant flavocytochrome 7 2 in E. call (Black et al., 1989) has allowed the active site of the enzyme to be probed using site-directed mutagenesis. Two particular residues, His373 and Tyr254, have been examined in detail, since they have important roles in catalysis. The substitution of His373 by glutamine resulted in an enzyme with some 

FIGURE 4. Mechanisms for lactate dehydrogenation, a. By hydride transfer His373 abstracts the hydroxyl proton and promotes hydride transfer to the N-5 of the flavin, b. By carbanion formation His373 removes the OG-carbon hydrogen as a proton forming a carbanion. A subsequent transfer of two-electrons to the flavin is required. 

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