Uncovering novel heme chemistry in microbial systems

Project Summary In nature, metalloenzymes make up approximately one third of the known enzymes and play central roles in biological processes such as photosynthesis, respiration, and nitrogen fixation. Heme-containing metalloenzymes are particularly important in the context of energy conversion reactions due in part to their ability

to cycle between redox states. Furthermore, heme-containing enzymes can oxidize a broad range of substrates in both a stereospecific and regiospecific manner, attracting interest for use in challenging chemical reactions requiring C-H activation, C-C bond cleavage, or heteroatom oxidation. These enzymes are thus prime candidates

for biotechnological and synthetic applications, including production of pharmaceutical drugs, chemical conversion of fatty acids and steroids, detoxification of drugs and toxins, and the synthesis of the industrially relevant chemicals, fragrances, and flavors. While heme proteins have been studied extensively, increasing amounts of genomic data have led to the

prediction of many additional heme, diheme, and multiheme protein families, of which the functional roles and biochemical properties remain unexplored. Once such protein is MbnH, originally identified in methanotrophic bacteria. MbnH is a member of the PF03150 bacterial cytochrome c peroxidase (bCcP)/MauG superfamily and

utilizes a rare bis-FeIV cofactor for the oxidation of a specific tryptophan residue within a partner protein, MbnP. While other high-valent Fe states, such as compound I and compound ES, are relatively well understood, given the limited biological examples of diheme bis-FeIV states, there is a fundamental gap in our understanding of

how nature stabilizes and uses bis-FeIV states without causing cellular damage. Nearly all previous research has focused on MauG, limiting the generalizability of the role of the bis-FeIV species and the chemical capacity of such an oxidant. Thus, the proposed research will leverage the huge amount of genomic information available

to expand the scope of studies on the bCcP/MauG superfamily. This includes representative members of the annotated but uncharacterized protein families, SPOA0271 and TIGR3891, as well as new cluster identified by the Enzyme Function Initiative Genome-Neighborhood Tool (EFI-GNT) tool as clusters 4 and 11. The broad

objective of this application is to establish how divergent bacterial diheme proteins stabilize high-valent bis-FeIV states and then use that intermediates to accomplish site-specific substrate oxidation. It will address outstanding questions related to the formation and stabilization of bis-FeIV, the catalytic capability of the bis-FeIV species, and

the biological role of this cofactor.