Tuesday, September 5, 2023 - 2:00pm
Abstract: Cytochromes P450 (P450s) are heme-containing monooxygenases that catalyze a variety of reactions, such as hydroxylation, epoxidation, and C–C bond breakage. Significant efforts have gone into the study of the catalytic cycle, in which high-valent intermediates are generated under physiological conditions to break unactivated C–H bonds. The various forms of regulation of the catalytic cycle required to ensure catalysis proceeds only under the appropriate physiological conditions are diverse and less well understood. For example, regulation is important in order to avoid wasting otherwise valuable reducing equivalents, preventing high-valent intermediates from degrading the protein, and/or preventing the release of reactive oxygen species. This thesis focuses on a few regulatory aspects of the catalytic cycle. One such aspect is substrate binding in a bacterial P450, named P450terp, after the substrate α-terpineol. The crystal structure of P450terp reveals a second substrate binding site. Substrate binding studies reveal that this second binding site is important for binding of substrate in the first binding site and turnover assays show that the second substrate binding site is necessary for efficient catalysis. Another aspect of regulation is the binding of the protein redox partner, in what has historically been called the effector role. I focus on Pseudomonad P450s, since their similar biological roles might lead to similar structure function relationships. Studies on P450terp and P450lin, named after the substrate linalool, reveal that they are promiscuous, able to turnover substrate with an exogenous protein redox partner. While this marks a difference from the model Pseudomonad system, there is a consistency in the effector role of the protein redox partner that is shared by many P450s. In all P450s that utilize a protein redox partner studied thus far, binding of the redox partner results in structural changes in the P450, as evidenced by an increase in the decay of an intermediate in the catalytic cycle called the oxycomplex. Besides these two external forms of regulation, I also look into the effects of mutating residues in the active site. One such example is mutation of the residue immediately following the cysteine residue ligating the heme in an investigation into a “proximal push” that might mimic redox partner binding and possible effects on the stability of high-valent intermediates in the catalytic cycle. Another example is the mutation of certain residues in an investigation of the origin of the P420 species, which is considered to be an inactivated, damaged P450 incapable of performing catalysis. Together, all of these studies have advanced our understanding of regulation of the P450 catalytic cycle.


Jessica Gable


NS2 1201