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-catalyzedl-lactate oxidation reaction, a still-debated issue. In the calculated enzyme-substrate model complex, thel-lactate α-OH hydrogen is hydrogen bonded to the active-site base H373 Nε, whereas the Hα is directed towards flavin N5, suggesting that the reaction is initiated by α-OH proton abstraction. Starting from this structure, simulation ofl-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 α-OH proton abstraction preceding Hα 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