Heme enzymes perform important transformations such as C-C bond cleavage, heteroatom oxidation, and C-H activation.1 Here, we address an emerging class of bacterial diheme enzymes that utilize an unusually stable, high-valent, bis-FeIV state for oxidative transformations.2,3 A recently identified member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, MbnH, was discovered to use a high-valent iron intermediate to cleave the bond between the Nε1 and Cδ1 atoms of the indole ring of a conserved tryptophan residue on its partner protein, MbnP.4,5 This modification is unique in that the installation of kynurenine by MbnH combines the activities of tryptophan-2,3-dioxygenase and N-formyl kynurenine formamidase, thereby representing new diheme chemistry.1 However, the mechanism by which MbnH catalyzes this reaction and how it deploys such oxidizing equivalents without causing self-oxidative damage is currently unknown.
In this work, we use MbnP peptide analogues to investigate the substrate interaction with MbnH by ITC, SPR, and x-ray crystallography. We also report a new bCcP/MauG superfamily protein, VMPH, containing domains with sequence similarities to MbnH and MbnP thus providing insight into substrate interaction. To assess how MbnH may avoid oxidative damage in the absence of substrate, select tyrosine mutants potentially involved in the transport of oxidizing equivalents were examined using UV-Vis-nIR and stopped-flow spectroscopy.6 We also perform rapid freeze-quench and electron paramagnetic resonance to view transient reaction intermediates formed during this decay to elucidate how MbnH moves oxidizing equivalents from the diheme core. The results presented here provide insight into the mechanism of oxidative modification performed by this family of diheme enzymes and more broadly suggests a means by which nature handles and deploys potent oxidizing intermediates.