Further evidence in favour of a carbanion mechanism for glycolate oxidase.

The flavoenzyme glycolate oxidase oxidizes glycolic acid to glyoxylate and the latter, more slowly, to oxalate. It is a member of an FMN-dependent enzyme family that oxidizes l-2-hydroxy acids to keto acids. There has been a controversy concerning the chemical mechanism of substrate oxidation by these enzymes. Do they proceed by hydride transfer, as observed for NAD-dependent enzymes, or by initial formation of a carbanion that transfers the electrons to the flavin? The present work describes a comparison of the reactivity of glycolate, lactate and trifluorolactate with recombinant human glycolate oxidase, by means of rapid-kinetics experiments in anaerobiosis. We show that trifluorolactate is a substrate for glycolate oxidase, whereas it is known as an inhibitor for NAD-dependent enzymes, as is trifluoroethanol for NAD-dependent alcohol dehydrogenases. Unexpectedly, it was observed that, once reduced, a flavin transfers an electron to an oxidized flavin, so that the end species is a flavin semiquinone, whatever the substrate. This phenomenon has not previously been described for a glycolate oxidase. Altogether, considering that another member of this flavoenzyme family (flavocytochrome b , a lactate dehydrogenase) has also been shown to oxidize trifluorolactate (Lederer F et al. (2016) Biochim Biophys Acta 1864, 1215-21), this work provides another important piece of evidence which is hardly compatible with a hydride transfer mechanism for this flavoenzyme family.