l‐Lactate dehydrogenation in flavocytochrome b₂

Abstract

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 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 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 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.

QM/MM study ofl-lactate oxidation by flavocytochrome b2

“…55 The final product with pyruvate is less stable than reactants, suggesting that it is less stabilized in the active site. These results are coherent with those previously obtained by Tabacchi et al 28 and the fact that no solvent isotope effect was encountered in this system experimentally. 26 The other pathway (yellow line in Figure 4B) consists in simultaneous proton and hydride transfers and is associated with a free energy of activation of 29.8 kcal/mol.…”

“…A first computational study was published by Tabacchi et al 28 occurs via a concerted but asy hro ous m ha sm wh r th α-hydroxyl proton transfer to H373 precedes the hydride transfer to FMN assisted by the isoalloxazine C4α-C α " tropav d path". 31 The reported activation free energy value was 12.1 kcal/mol which was compatible with the estimated experimental value of 13.5-13.6 kcal/mol.…”

Lactate oxidation facilitates growth of Mycobacterium tuberculosis in human macrophages

“…Nonetheless, the highly conserved active site strongly implies that LldD1 is involved in the oxidation of small α-hydroxy acids such as lactate 19,20 . We therefore determined if LldD1 is required for the oxidation of other α-hydroxy acids.…”

Oxidation of amines by flavoproteins

“…The flavoproteins that oxidize α-hydroxy acids include glycolate oxidase, lactate monooxygenase, flavocytochrome b 2 , and mandelate dehydrogenase and make up a separate structural family [133–136]. Although their mechanism of oxidation has been controversial [137, 138], structural, mechanistic, and computational studies support a mechanism similar to that of the alcohol oxidases, hydride transfer from the alkoxide form of the substrate [139–141]. …”