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.

While ubiquitous in the biological realm, heme enzymes exhibit numerous functions, including protecting organisms against reactive oxygen species, detoxifying xenobiotics and neuronal signaling. The specific function of a heme enzyme is tuned by the structure of the protein that binds the prosthetic group. This relationship between heme and protein is why heme enzymes have long served a foundational role in probing structure-function relationships in biology. The aim of this dissertation is to gain additional insights into heme enzyme structure-function relationships, as well as to gain a better understanding of heme enzyme active site chemistry and the interactions between the enzymes and their redox partners. We have done this by studying four different heme enzyme systems: i) a cytochrome c peroxidase from Leishmania major dubbed Leishmania major Peroxidase (LmP), ii) a speculative peroxynitrite isomerase, also from L. major dubbed pseudoperoxidase (LmPP), iii) a cytochrome P450 monooxygenase from Citrobacter braakii called P450cin, and iv) several mammalian nitric oxide synthase (NOS) isoforms. We determined the precise catalytic mechanism LmP, including the intriguing rate-limiting step of catalysis at steady-state and the importance of proton transfer in the catalytic cycle. We also captured the X-ray crystal structure of the first catalytic intermediate with an iron center unreduced by electrons. This pristine structure, taken together with kinetics, corroborates our mechanistic conclusions. In the process, we validated the usefulness of X-ray free electron lasers as a powerful tool to probe the structure of enzymes that contain transition metals. We probed the association of LmP with its redox partner LmCytc and discovered the existence of a non-catalytic binding site. For our second system, we have solved the crystal structure of a novel LmPP and proposed a hypothetical mechanism that awaits testing. Although we were unable to reach our goals for our third system, we established a protocol to address the crystallization of the P450cin-Cdx complex. For our fourth system, we explored the precise and sensitive balance of forces that stabilize the structure and thus function of NOS, which is maintained by an elegant synergistic relationship between the Zn2+, cofactor, and substrate binding sites. We finish by describing collaborative work attempting to design potent, isomer selective and bioavailable inhibitors to treat neurodegenerative diseases.