Abstract
Recently, the discovery of a novel Mn/Fe protein in a number of pathogens and extremophiles has led to the establishment of a new group of proteins, dubbed R2lox (R2‐like ligand‐binding oxidase). Originally flagged for being highly upregulated in the virulent strain of Mycobacterium tuberculosis, R2lox was initially identified as a sequence homolog for the R2 subunit in class 1 ribonucleotide reductase (RNR); however, a lack of RNR activity combined with x‐ray crystallography data indicate significant differences between R2lox and the typical RNRs. Despite conventional inorganic wisdom and the presence of identical ligands, R2lox has been shown in vitro to bind MnII in the presence of FeII and spontaneously self‐assembles with a site‐differentiated Mn/Fe cofactor. In the absence of MnII, a canonical Fe/Fe cofactor assembles. More interestingly, upon O2 exposure, both cofactors oxidize a nearby valine residue, forming an unprecedented tyrosine‐valine crosslink via C‐H bond activation. However, work in our lab has indicated the mechanisms by which these two cofactors perform this chemistry is thoroughly distinct. It is our goal to utilize the R2lox system as a model to further explore factors governing metallocofactor formation, as well as investigate the potential of these cofactors to perform challenging substrate oxidation reactions using divergent pathways. Utilizing aerobic UV/vis, stopped‐flow, and EPR spectroscopies, the kinetics of metal binding, cofactor assembly, and characterization of discrete intermediates were studied in both Mn/Fe and Fe/Fe R2lox. Through this work, a number of distinctions between the mechanisms for Mn/Fe and Fe/Fe cofactor maturation were identified, and models for the assembly processes have been developed. The implications of these findings are further discussed in the context of potential relevance of R2lox towards therapeutic development and biocatalysis.Support or Funding InformationThe Ohio State University, Pelotonia Fellowship Program, and NIH Cellular, Molecular, and Biochemical Sciences Training Grant
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