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

The diheme 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 toward H2O2 and the O-2 analogue CO was essentially the same as that of MauG in the preformed enzyme-substrate complex. The addition of H2O2 to diferric MauG generated a diheme bis-Fe(TV) species [i.e., Fe(IV)=O/Fe(IV)] which formed at a rate of >300 s(-1) and spontaneously returned to the diferric state at a rate of 2 x 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 K-d of <= 1.5 mu M 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 preformed enzyme-substrate complex to generate bis-Fe(IV) MauG followed by reaction with PreMADH. Stopped-flow kinetic studies of the reaction of diferrous MauG with CO yielded a faster major transition with a bimolecular rate constant of 5.4 x 10(5) M-1 s(-1), and slower transition with a rate of 16 s(-1) which was independent of CO concentration. The same rates were obtained for binding of CO to diferrous 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 toward oxygen in the absence of substrate. This kinetic mechanism is also distinct from that of the homologous diheme cytochrome c peroxidases that require a mixed valence state for activity.