L-lactate dehydrogenation in flavocytochrome b2: a first principles molecular dynamics study.

First principles molecular dynamics studies on active-site models of flavocytochrome b2 (L-lactate : cytochrome c oxidoreductase, Fcb2), in complex with the substrate, were carried out for the first time to contribute towards establishing the mechanism of the enzyme-catalyzed L-lactate oxidation reaction, a still-debated issue. In the calculated enzyme-substrate model complex, the L-lactate alpha-OH hydrogen is hydrogen bonded to the active-site base H373 Nepsilon, whereas the Halpha is directed towards flavin N5, suggesting that the reaction is initiated by alpha-OH proton abstraction. Starting from this structure, simulation of L-lactate oxidation led to formation of the reduced enzyme-pyruvate complex by transfer of a hydride from lactate to flavin mononucleotide, without intermediates, but with alpha-OH proton abstraction preceding Halpha transfer and a calculated free energy barrier (12.1 kcal mol(-1)) consistent with that determined experimentally (13.5 kcal mol(-1)). Simulation results also revealed features that are of relevance to the understanding of catalysis in Fcb2 homologs and in a number of flavoenzymes. Namely, they highlighted the role of: (a) the flavin mononucleotide-ribityl chain 2'OH group in maintaining the conserved K349 in a geometry favoring flavin reduction; (b) an active site water molecule belonging to a S371-Wat-D282-H373 hydrogen-bonded chain, conserved in the structures of Fcb2 family members, which modulates the reactivity of the key catalytic histidine; and (c) the flavin C4a-C10a locus in facilitating proton transfer from the substrate to the active-site base, favoring the initial step of the lactate dehydrogenation reaction.