Heme and Protein Radicals

A distinguishing trait of heme enzymes is that a high-valent iron-oxo species is the common oxidant for mediating a remarkable array of oxidation reactions. However, the conundrum is that each enzyme in general only promotes a specific type of reaction. How the reaction type is determined after formation of the key oxidant remains to be an unsolved question. Answers to this question have implications for our fundamental understanding of enzyme catalysis as well as de novo enzyme design and protein engineering. This application focuses on a mechanistic characterization of three tyrosine-oxidizing enzymes attempting to uncover the key factors governing hydroxylation of the organic substrate. Each of these enzymes employs a mononuclear heme cofactor to oxidize its tyrosine-based substrate. The peroxidases LmbB2 and SfmD promote efficient and selective hydroxylation of a tyrosine substrate. Intriguingly, the third enzyme, a cytochrome P450 protein CYP121 is expected to perform a hydroxylation reaction, however, it does an unusual non-hydroxylating oxidation reaction. Given the similarities of the heme-based oxidant and the structure of the substrates, the inevitable question arises regarding the factor that determines the catalytic activity in these enzymes. In Aim #1, we will determine the structural characteristics of the enzyme active site, with emphasis on how the substrate is positioned to the iron-bound oxidant. Using a battery of synthetic probes, we will also attempt to stall the coupling or the hydroxylation reactions to glean insight into their respective mechanisms. In Aim #2, we will characterize the contribution of the axial ligand and the second coordination sphere to the reactivity of the heme oxidant during catalysis in these enzymes. The results obtained from the in-depth analysis of these three related catalytic systems will unravel the structure-function relationships of the heme enzymes and address the mechanism of substituted phenol hydroxylation.