UC Irvine

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.