title: Kinetic mechanism for the initial steps in MauG-dependent tryptophan tryptophylquinone biosynthesis.
abstract: 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 H(2)O(2) and the O(2) analogue CO was essentially the same as that of MauG in the preformed enzyme-substrate complex. The addition of H(2)O(2) to diferric MauG generated a diheme bis-Fe(IV) 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 < or = 1.5 microM for the MauG-PreMADH complex. The results were the same whether bis-Fe(IV) MauG was mixed with PreMADH or H(2)O(2) 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.
authors.affiliation: Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
authors.forename: Sheeyong
authors.initials: S
authors.lastname: Lee
authors.forename: Sooim
authors.initials: S
authors.lastname: Shin
authors.forename: Xianghui
authors.initials: X
authors.lastname: Li
authors.forename: Victor L
authors.initials: VL
authors.lastname: Davidson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D045563:Indolequinones / Q000096:biosynthesis*; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D045563:Indolequinones; C070569:tryptophan tryptophylquinone; D014364:Tryptophan; D006861:Hydrogen Peroxide; D010100:Oxygen