As such, the steady state kinetic reaction of MauG-dependent TTQ formation from quinol MADH was studied and compared to the steady state reaction with preMADH as the substrate for TTQ biosynthesis ( Scheme 1). Quinol MADH was not oxidized by H 2O 2 alone, but in the presence of MauG, H 2O 2-dependent oxidation of the quinol to TTQ was observed suggesting that this might be the final step in TTQ biosynthesis in vivo. To test this hypothesis, the quinol was formed in vitro by reduction of MADH with dithionite and then tested as a substrate for MauG. The quinol was postulated to be an intermediate in TTQ biosynthesis from preMADH because a transient intermediate with a λ max at 330 nm was observed early in the steady-state TTQ biosynthesis reaction using preMADH as a substrate ( 9). The fully oxidized quinone exhibits a broad absorbance centered at 440 nm, the one-electron reduced semiquinone exhibits an absorption maximum at 428 nm, and the fully reduced quinol exhibits an absorption maximum at 330 nm ( 16). While the order is not known, the overall process of MauG-dependent TTQ biosynthesis requires three sequential two-electron oxidations to catalyze the second hydroxylation of βTrp57, the crosslink formation between βTrp57 and βTrp108, and the oxidation to the quinone state.Īs TTQ is a two-electron redox cofactor, it may be present in MADH in three different redox states. The sole axial ligand for the ferryl heme is His35, and the axial ligands for the other heme are His205 and Tyr294 ( 15). The TTQ biosynthetic reactions proceed via a relatively stable high valent bis-Fe(IV) intermediate with one heme as Fe(IV)=O and the other as Fe(IV) with two axial ligands from the protein ( 14). Incubation of preMADH in vitro with MauG in the presence of either O 2 plus an electron donor or H 2O 2 as oxidizing equivalents results in completion of TTQ biosynthesis and active MADH ( 5, 9). Inactivation of mauG, a gene in the methylamine utilization ( mau) gene cluster ( 11) leads to accumulation of a biosynthetic precursor protein (preMADH) in which βTrp57 is monohydroxylated at C7 position, and the covalent crosslink between βTrp57 and βTrp108 is absent ( 12, 13). MauG exhibits homology to diheme cytochrome c peroxidase ( 7, 8), but displays significant differences in catalytic and redox behavior ( 9, 10). TTQ biosynthesis requires the action of MauG ( 5) which is a 42.3 kDa enzyme containing two c-type hemes ( 6). For TTQ biosynthesis, two atoms of oxygen are incorporated into the indole ring of βTrp57 and a covalent bond is formed between the indole rings of βTrp57 and βTrp108. TTQ is formed by post-translational modification of two tryptophan residues of the polypeptide chain. Methylamine dehydrogenase (MADH) 1 is a 119 kDa heterotetrameric α 2β 2 protein ( 1, 2) with each β subunit possessing a tryptophan tryptophylquinone (TTQ) ( 3) protein-derived cofactor ( 4). Analysis of the structure of the MauG-preMADH complex in the context of ET theory and these results suggests that direct electron tunneling between the residues which form TTQ and the five-coordinate oxygen-binding heme is not possible, and that ET requires electron hopping via the six-coordinate heme. The difference in rate constants is consistent with a larger driving force for the faster reaction. These similar K d values are much greater than that for the MauG-preMADH complex, indicating that the extent of TTQ maturity rather than its redox state influences complex formation. ET from diferrous MauG to oxidized TTQ of MADH exhibits a K d of 10.1 μM and rate constant of 0.07 s −1. Interprotein ET from quinol MADH to the high valent bis-Fe(IV) form of MauG exhibits a K d of 11.2 μM and a rate constant of 20 s −1. The kinetics of two ET reactions between MADH and MauG have been analyzed. This six-electron oxidation of preMADH requires long range electron transfer (ET) as the structure of the MauG-preMADH complex reveals that the shortest distance between the modified residues of preMADH and the nearest heme of MauG is 14.0 Å. The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor.
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