Photochemical Oxidation of Wild Type and Mutant Cytochrome P450 CAM

Abstract

The goal of my research project is to study the role of tryrosines 29 and 96 in the oxidation-reduction chemistry of P450 CAM cytochromes: ubiquitous heme-thiolate proteins. P450 proteins were discovered in 1958 by their unusual reduced carbon monoxide difference spectrum that has an absorbance at 450 nm, hence Pigment at 450 or P450. This odd spectrum is derived from a thiolate anion acting as the 5th ligand to the heme. In this experiment, we used light to oxidize the protein. The significance of tyrosines 29 and 96 is that they are located adjacent to theheme group. Other than the iron in the heme, the likely candidate for an oxidation reaction within the protein is a tyrosine residue because it has the lowest oxidation potential of the amino acids. Furthermore, prior work has shown that if any amino acid is indeed going to be oxidized in this photochemical experiment, it will be a tyrosine. In order to study the role that tyrosines 29 and 96 play in the oxidation-reduction of P450 cytochromes, wemutated those tyrosines to phenylalanines, which have very similar structures to tyrosines and this should be the least perturbing to the global structure of the protein. Through examining the shift in the mutant P450's redox potential we hope to gain a greater understanding of tyrosines 29 and 96's importance in electron transport.