Figure 4. Proposed electron transfer pathways in MbnH. Amino acid residues that comprise different possible pathways are shown in color.

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. One such protein is MbnH, a divergent member of cytochrome c peroxidase (bCcP)/MauG superfamily (PF03150, Figure 1), which uses a bis-Fe^IV species involving two hemes and an intervening tryptophan (Figure 2) to install a kynurenine in a partner protein. 

 A central hypothesis of this research project is that this superfamily represents a largely unexplored new class of metalloenzymes that, like MbnH, store a second oxidizing equivalent on the iron of a second heme, creating an extended conjugated system that displays unusual kinetic stability (Figure 3). This type of system challenges the notion long held by chemists that very potent, high-valent, oxidizing species are “hot” intermediates with short lifetimes and rapid decay rates. However, despite the existence of over 18,000 unique family members (Figure 1), nearly all previous research has focused on only one protein, MauG, limiting the generalizability of the role of the bis-Fe^IV species. Thus, leveraging the huge amount of genomic information available, the proposed research will expand the scope of studies on the bCcP/MauG superfamily to include unknown protein families identified by the Enzyme Function Initiative Genome-Neighborhood Tool (EFI-GNT) as distinct clusters within the broad superfamily (Figure 1). Combining the bioinformatics analyses with the tools of structural biology, spectroscopy, biochemistry, and microbiology, we will assess selected family members for their capacity to utilize a bis-Fe^IV state for challenging oxidation reactions and biological relevance of such reactions.

This project will focus on the guiding questions:

Is the intervening tryptophan between the two hemes required for cofactor formation?
What is the role of tyrosine in bis-Fe^IV formation?
What does the process of substrate recognition look like?
Are all substrates partner proteins? 
Can we also modify peptides or designed peptides, or is this protein specific? 
What makes the bis-Fe^IV so kinetically stable? 
What residues in the heme pocket stabilize bis-Fe^IV vs. other high-valent states? 
How do these proteins protect themselves from oxidative damage?
Do bis-Fe^IV  forming enzymes only modify tryptophans or can they modify other amino acids?
Can we use these enzymes or their products to expand our enzyme toolbox for biocatalysis?
Can we design artificial bis-Fe^IV forming enzymes?

Proteins are produced by all living organisms and generally are critical components in all cellular processes. In order to be active, most proteins need to assemble into a three-dimensional structure that is encoded by its amino acid sequence. Generally, proteins are comprised of a combination of only 20 canonical amino acids, which then limits the structural, chemical, and functional space occupied by the amino acid side chain groups. Combined with a limited set of bioavailable metals, one may wonder how nature creates structurally novel proteins with diverse functions and metal-binding sites. We think one way biological systems overcome this limitation is through the introduction of post-translational modifications to amino acids which can often result in new functionality, structures, or catalytic capability.

Specifically, tryptophan is an important metabolic precursor for many signaling molecules in eukaryotes, plants, and bacteria. Its transformation as a free metabolite has been well documented in eukaryotic systems however, how tryptophan gets modified in bacterial systems and the chemical scope of those modifications is less well-understood. To date, the biochemical significance of kynurenine has only been studied within the context of metabolism, as 95% of tryptophan is catabolized through the kynurenine pathway. Beyond tryptophan metabolism, there are limited studies on kynurenine as a metal-binding residue. The first kynurenine-containing proteins were identified in microbial species (Figure 5), in which modification to kynurenine results in the creation of a new copper-binding site. The function of this new metal site, as well as the enzymatic mechanism for how tryptophan gets modified to kynurenine remains unknown. 

This project will focus on the guiding questions of:

Can we use genomic neighborhood information, sequence information, and pattern recognition to predict which proteins will contain Kyn as a post-translational modification?
How is Kyn installed?
What is the biological role of Kyn?
Are Kyn-containing proteins only associated with Cu-binding or can those metal sites bind other metals?
How does Kyn influence the electronic structure of the metal site?
Are Kyn-containing proteins enzymes? If so, what chemistry might they catalyze? 
Do any of these Trp modifications result in interesting or bioactive natural products?