Mutation of Trp⁹³ of MauG to tyrosine causes loss of bound Ca²⁺ and alters the kinetic mechanism of tryptophan tryptophylquinone cofactor biosynthesis

The dihaem enzyme MauG catalyses a six-electron oxidation required for post-translational modification of preMADH (precursor of methylamine dehydrogenase) to complete the biosynthesis of its TTQ (tryptophan tryptophylquinone) cofactor. Trp⁹³ of MauG is positioned midway between its two haems, and in close proximity to a Ca²⁺ that is critical for MauG function. Mutation of Trp⁹³ to tyrosine caused loss of bound Ca²⁺ and changes in spectral features similar to those observed after removal of Ca²⁺ from WT (wild-type) MauG. However, whereas Ca²⁺-depleted WT MauG is inactive, W93Y MauG exhibited TTQ biosynthesis activity. The rate of TTQ biosynthesis from preMADH was much lower than that of WT MauG and exhibited highly unusual kinetic behaviour. The steady-state reaction exhibited a long lag phase, the duration of which was dependent on the concentration of preMADH. The accumulation of reaction intermediates, including a diradical species of preMADH and quinol MADH (methylamine dehydrogenase), was detected during this pre-steady-state phase. In contrast, steady-state oxidation of quinol MADH to TTQ, the final step of TTQ biosynthesis, exhibited no lag phase. A kinetic model is presented to explain the long pre-steady-state phase of the reaction of W93Y MauG, and the role of this conserved tryptophan residue in MauG and related dihaem enzymes is discussed.