Heme enzymes are ubiquitous throughout nature with diverse functions including C-H bond hydroxylation, C-C bond cleavage, and heteroatom oxidation. Canonical oxidative heme chemistry involves the formation of high-valent iron where one oxidizing equivalent is stored on the heme iron and the second oxidizing equivalent is stored as an organic radical either on the porphyrin ring/axial ligand (Compound I) or on a nearby amino acid residue (compound ES). A unique form of high-valent heme iron, originally discovered in the diheme enzyme MauG, stores the second oxidizing equivalent on the iron of a second heme rather than as an organic radical thereby forming a bis-FeIV state.1,2 To date, there are three known bis-FeIV forming enzymes each of which share oxidizing equivalents across two spatially distinct cofactors (~16 Å), yielding two high-valent iron sites associated with a transient feature in the near-IR region.3,4 Despite various spectroscopic investigations of the bis-FeIV state, a fundamental understanding of the electronic and magnetic properties is still lacking, and the physical origins of the near-IR transition remain unclear. Moreover, there remains a gap in our understanding of how nature forms and stabilizes the bis-FeIV state.
Here, we performed site-directed mutagenesis on a key tryptophan residue located approximately halfway between the two heme sites in the bis-FeIV forming enzyme, MbnH to assess its necessity in bis-FeIV formation and decay. Using electron paramagnetic resonance and resonance Raman, we have developed an emerging picture of the electronic structure of diferric resting and bis-FeIV states of MbnH. Coupled with kinetic analysis and isotopic substitution we further find that bis-FeIV formation is not dependent on the intervening Trp and decay of the high-valent state is proton-dependent. Together, these results provide insight into key spectroscopic metrics for bis-FeIV forming enzymes and provide an alternative mechanism for how nature may store oxidizing equivalents.