Carboxyl Group of Glu113 Is Required for Stabilization of the Diferrous and Bis-Fe^IV States of MauG

Abstract

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies have implicated Glu113 in the formation of the bis-FeIV state of MauG, in which one heme is FeIV=O and the other is FeIV with His-Tyr axial ligation. An E113Q mutation had no effect on the structure of MauG, but significantly altered its redox properties. E113Q MauG could not be converted to the diferrous state by reduction with dithionite, but was only reduced to a mixed valence FeII/FeIII state, which is never observed in wild-type (WT) MauG. Addition of H2O2 to E113Q MauG generated a high valence state that formed more slowly and was less stable than the bis-FeIV state of WT MauG. E113Q MauG exhibited no detectable TTQ biosynthesis activity in a steady-state assay with preMADH as the substrate. It did catalyze the steady-state oxidation of quinol MADH to the quinone, but 1000-fold less efficiently than WT MauG. Addition of H2O2 to a crystal of the E113Q MauG-preMADH complex resulted in partial synthesis of TTQ. Extended exposure of these crystals to H2O2 resulted in hydroxylation of Pro107 in the distal pocket of the high-spin heme. It is concluded that the loss of the carboxylic group of Glu113 disrupts the redox cooperativity between hemes that allows rapid formation of the diferrous state, and alters the distribution of high-valence species that participate in charge-resonance stabilization of the bis-FeIV redox state.

Roles of multiple-proton transfer pathways and proton-coupled electron transfer in the reactivity of the bis -Fe IV state of MauG

“…The bis-Fe IV state of MauG describes an alternative strategy by which to stabilize a high-valent heme iron. Several amino acid residues in the proximity of the two hemes have also been implicated in facilitating the formation and stabilizing the bis-Fe IV state (21,(28)(29)(30)(31)(32). The concept of CR stabilization of this high-valent state led to the description of it actually comprising an ensemble of resonance structures including the more traditional high-valent heme species, but with the true bis-Fe IV state as the dominant species (6, 9) (Fig.…”

“…E113Q and Q103N mutations each affected the stability and the rate of the spontaneous decay of the high-valent state. An E113Q mutation disturbed the hydrogen bonding network from the ferryl oxygen to Asn110 (28). A Q103N mutation altered the water network in that an additional ordered water is present in the place of the amide nitrogen of Gln103 (30).…”

A T67A mutation in the proximal pocket of the high-spin heme of MauG stabilizes formation of a mixed-valent FeII/FeIII state and enhances charge resonance stabilization of the bis-FeIV state

“…A mixed-valent state was not observed for WT MauG, or all but one of the many other variant MauG proteins which have been previously studied [7, 10, 17, 18, 22, 26]. The exception is E113Q MauG, which like T67A MauG could only be reduced by dithionite to a mixed-valent state which exhibited an absorbance spectrum very similar to that of T67A MauG [25]. …”

“…It is noteworthy that E113Q MauG, which also could only be reduced to the mixed valent state, also yielded a single E m value. However, that value was −196 mV [25]. …”

“…Mutation of Gln103 significantly affected the overall protein stability of MauG and altered the redox properties of the hemes [24]. The carboxyl group of Glu113 was shown to be an important determinant of the distribution of high-valent species that participate in charge-resonance stabilization of the bis -Fe IV redox state, as well as in the maintenance of the redox cooperativity between the two hemes of MauG [25]. …”