Deciphering the Two-Step Hydride Mechanism of Monoamine Oxidase Flavoenzymes

The complete two-step hydride transfer mechanism of amine
oxidation
involved in the metabolism of monoamine neurotransmitters was scrutinized
by DFT calculations. In living organisms, this process is catalyzed
by monoamine oxidase enzymes. Herein, we focus on some intriguing
aspects of the reaction that may have been previously noticed but
have not been clarified to date. The first step of the reaction includes
the C–H bond cleavage on the methylene group vicinal to the
amino group of the monoamine substrate and the subsequent transfer
of hydrogen to the N5 atom of the flavin prosthetic group of the enzyme.
We confirmed the nature of this step to be hydride transfer by evaluation
of the pertinent HOMO–LUMO gap together with analysis of orbital
contours alongside the intrinsic reaction coordinate profile. Next,
we investigated the rather peculiar intermediate adduct that may form
between the amine substrate and the flavin molecule, featuring an
unusually long C–N bond of ∼1.62 Å. Although this
bond is quite stable in the gas phase, the presence of just a few
explicit water molecules facilitates its dissociation almost without
energy input so that the amine-flavin intermediate can form an ionic
pair instead. We attribute the existence of the unusual C–N
bond to a fragile balance between opposing electronic structure effects,
as evaluated by the natural bond orbital analysis. In line with this,
the intermediate in the solution or in the enzyme active site can
exist in two energetically almost equivalent forms, namely, as a covalently
bound complex or as an ion pair, as suggested by previous studies.
Finally, we characterized the transformation of the intermediate to
the fully reduced flavin and imine products via proton transfer from
the amino group to the flavin N1 atom, completing the reductive part
of the catalytic cycle. Although we found that explicit solvation
substantially boosts the kinetics of this step, the corresponding
barrier is significantly lower than that in the hydride transfer step,
confirming hydrogen abstraction as the rate-limiting step of amine
oxidation and validating the two-step hydride transfer mechanism of
monoamine oxidases.