Flavocytochrome b2 catalyses the oxidation of L-lactate to pyruvate in yeast mitochondrial intermembrane space. Its flavoprotein domain is a member of a family of FMN-dependent 2-hydroxy-acid-oxidizing enzymes. Numerous solution studies suggest that the first step of the reaction consists of proton abstraction from lactate C2, leading to a carbanion that subsequently yields electrons to FMN. The crystal structure suggests that the enzyme base is His373, and that Tyr254 may be hydrogen bonded to the substrate hydroxyl. Studies carried out with the Y254F mutant [Dubois, J., Chapman, S.K., Mathews, F.S., Reid, G.A. & Lederer, F. (1990) Biochemistry 29, 6393-6400] showed that Tyr254 does not act as a base but stabilizes the transition state. As the mutation did not induce any change in substrate affinity, the question of the existence of the hydrogen bond in the Michaelis complex remained open. Similar results with glycolate oxidase, mutated at the same position, led to the suggestion that these enzymes actually operate via a hydride transfer mechanism [Macheroux, P., Kieweg, V., Massey, V., Soderlind, E., Stenberg, K. & Lindqvist, Y. (1993) Eur. J. Biochem. 213, 1047-1054]. In the present work, we have re-investigated the matter by analysing the properties of a Y254L mutant flavocytochrome b2, as well as the behaviour of the Y254F enzyme with two substrates other than lactate, and a series of inhibitors. The Y254L protein is less efficient with L-lactate than the wild-type enzyme by a factor of 500, but the substrate affinity is unchanged. In contrast, L-phenyllactate and mandelate, poor substrates (the latter acting more as an inhibitor), exhibit an increased affinity. In addition, the Y254L mutant enzyme is more efficient with phenyllactate than lactate as a substrate. In order to rationalize these observations, we have modelled phenyllactate and mandelate in the active site, using previously described modelling experiments with lactate as a starting point. The results indicate that mandelate cannot bind in an orientation allowing proton abstraction by His373, due to steric interference by the side chains of Ala198 and Leu230. It might possibly adopt a binding mode as proposed previously for lactate, which leads to a hydride transfer and with which the 198 and 230 side chains do not interfere. However, other researchers [Sinclair, R., Reid, G.A. & Chapman, S.K. (1998) Biochem. J. 333, 117-120] showed that A198G, L230A and A198G/L230A mutant enzymes exhibit a strongly improved mandelate dehydrogenase activity. These results indicate that relief of the steric crowding facilitates catalysis by enabling a better mandelate orientation at the active site, suggesting that its productive binding mode is similar to that proposed for lactate in the carbanion mechanism. The modelling studies therefore support the hypothesis of a carbanion mechanism for all substrates. In addition, we present the effect of the two mutations at position 254 on the binding of a number of competitive inhibitors (such as sulfite, D-lactate, propionate) and of inhibitors that are known to bind at the active site both when the flavin is oxidized and when it is in the semiquinone state (propionate, oxalate and L-lactate at high concentrations). Unexpectedly, the results indicate that the integrity of Tyr254 is necessary for the binding of these inhibitors at the semiquinone stage.
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