Biosynthesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase in vitro.

摘要

Tryptophan tryptophylquinone (TTQ), the prosthetic group of methylamine dehydrogenase (MADH), is formed by post-translational modifications of two tryptophan residues that result in the incorporation of two oxygens into one tryptophan side chain and the covalent cross-linking of that side chain to a second tryptophan residue. MauG is a novel 42 kDa di-heme protein with low- and high-spin c-type hemes, which is required for the incorporation of the second oxygen and the cross-linking of the TTQ biosynthesis. The visible absorption and resonance Raman spectroscopic properties of each of the two c-type hemes, and the overall redox properties of MauG reveal that the two hemes with distinct spectral properties have similar intrinsic Em values but exhibit negative redox cooperativity. An experimental system has been developed that allows the direct continuous monitoring of MauG-dependent TTQ biosynthesis in vitro. Four diverse electron donors were shown to provide reducing equivalents for MauG-dependent TTQ biosynthesis under aerobic conditions. Peroxides and idosobenzenes could serve as alternative oxygen donors for MauG-dependent TTQ biosynthesis. During the reaction with H2O2, a discrete reaction intermediate was observed, which is likely the reduced quinol form of TTQ that is then oxidized to the quinone. The implications of these results in elucidating the mechanism of MauG-dependent TTQ biosynthesis are discussed.; Electron paramagnetic resonance (EPR) and visible absorption spectroscopy were used to identify intermediates in the MauG-dependent reactions. Addition of H2O2 to oxidized MauG results in a high-valent oxoferryl intermediate with the second oxidation equivalent located on the other heme iron (FeIV=O, FeIV). When the biological substrate of MauG, a biosynthetic precursor of MADH, is mixed with the high-valent oxoferryl intermediate a substrate-based radical intermediate is trapped. High-resolution size-exclusion chromatography shows that MauG can form a stable complex with the biosynthetic precursor of MADH and the high-valent intermediate, but not mature MADH. The maximum stoichiometry of binding of MauG to the precursor is 1:1. These findings reveal that significant conformational changes in one or both of the proteins occur during catalysis which significantly affects the protein-protein interactions. On the basis of these results, a mechanism of oxygen activation by MauG and a radical mechanism for MauG-dependent TTQ biosynthesis were proposed.