Kinetic Mechanism for the Initial Steps in MauG-dependent Tryptophan Tryptophylquinone Biosynthesis

摘要

The di-heme enzyme MauG catalyzes the biosynthesis of tryptophan tryptophylquinone (TTQ), the protein-derived cofactor of methylamine dehydrogenase (MADH). This process requires the six-electron oxidation of a 119 kDa MADH precursor protein with incompletely synthesized TTQ (PreMADH). The kinetic mechanism of the initial two-electron oxidation of this natural substrate by MauG was characterized. The relative reactivity of free MauG towards H2O2 and the O2 analog CO was essentially the same as that of MauG in the pre-formed enzyme-substrate complex. The addition of H2O2 to di-ferric MauG generated a di-heme bis-Fe(IV) species (i.e., Fe(IV)=O/Fe(IV)) which formed at a rate >300 s^-1 and spontaneously returned to the di-ferric state at a rate of 2×10^-4 s^-1 in the absence of substrate. The reaction of bis-Fe(IV) MauG with PreMADH exhibited saturation behavior with a limiting first-order rate constant of 0.8 s^-1 and a Kd ≤ 1.5 μM for the MauG-PreMADH complex. The results were the same whether bis-Fe(IV) MauG was mixed with PreMADH, or H2O2 was added to the pre-formed enzyme-substrate complex to generate bis-Fe(IV) MauG followed by reaction with PreMADH. Stopped-flow kinetic studies of the reaction of di-ferrous MauG with CO yielded a faster major transition with a bimolecular rate constant of 5.4 × 10⁵ M^-1s^-1, and slower transition with a rate 16 s^-1 which was independent of [CO]. The same rates were obtained for binding of CO to di-ferrous MauG in complex with PreMADH. This demonstration of a random kinetic mechanism for the first two-electron oxidation reaction of MauG-dependent TTQ biosynthesis, in which the order of addition of oxidizing equivalent and substrate does not matter, is atypical of those of heme-dependent oxygenases that are not generally reactive towards oxygen in the absence of substrate. This kinetic mechanism is also distinct from that of the homologous di-heme cytochrome c peroxidases that require a mixed valence state for activity.