Flavocytochrome carbanion mechanism

Flavin mononucleotide, 3absorption coefficients, 36 270 active site, 36 265-267 catalysis and electron transfer, 36 275-287 carbanion mechanism, 36 277-282 electron acceptors, 36 285-287 electron transfer pathway, 36 275-276, 282-285... 

Electron flow through flavocytochrome bz has been extensively studied in both the S. cerevisiae (Tegoni et al., 1998 Daff et al., 1996a Chapman et al., 1994 Pompon, 1980) and H. anomala (CapeillEre-Blandin et al., 1975) enzymes. The catalytic cycle is shown in Figure 3. Firstly, the flavin is reduced by L-lactate a carbanion mechanism has been proposed for this redox step (Lederer, 1991). Complete (two-electron) reduction of the flavin is followed by intra-molecular electron transfer from fully-reduced flavin to heme, generating flavin semiquinone and reduced heme (Daff et al.. 

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-... 

In the case of flavocytochrome 62 and related flavoenzymes, there is a sufficient body of evidence to indicate that the carbanion mechanism operates. The formation of a carbanion is not, of course, an oxidation and two electrons need to be transferred from the carbanion intermediate to the flavin cofactor. This could occur possibly via a covalent intermediate or by sequential one-electron transfers. These possibilities will be discussed in detail later in this section. 

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. 

Studies with the suicide inhibitor 2-hydroxy-3-butynoate (103, 113) have provided further support for a carbanion mechanism operating in flavocytochrome. The 2-hydroxy-3-butynoate results in a carbanion intermediate that can resonate to an allenic carbanion, which can then form a covalent adduct with the flavin cofactor (Fig. 11). This adduct is highly reactive and cyclizes to form an inactive modifled flavin group (113, 114). 

With D-amino acid oxidase, one of the diagnostic tests for a carbanion mechanism was that ethane nitronate acted as a substrate (110). However, with flavocytochrome 62 there is no evidence of electron transfer between ethane nitronate and the enzyme, rather this compound behaves as a competitive inhibitor (96). The reason why there is no electron transfer in the case of flavocytochrome 62 is unclear, but one possibility is that the carbanion is not correctly oriented. This absence of electron transfer in no way disproves a carbanion mechanism. Indeed, other flavoenzymes, such as long-chain hydroxyacid oxidase also fail to utilize ethane nitronate (115). So, apart from the ethane nitronate result, there is substantial evidence to support a carbanion mechanism in flavocytochrome 62 The question now is how do the electrons transfer to the oxidized FMN There are essentially three possibilities (1) there is a nucleophilic attack by the substrate carbanion at flavin N5, forming a covalent bond, with subsequent cleavage resulting in reduced flavin (2) there is a one-electron transfer to the flavin followed by collapse of the radical pair to again form a... 

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.

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