Nitric Oxide (NO) is involved in numerous physiological functions including vasodilatation.neurotransmission,and cytotoxic actions of the immune system. Understanding NO synthesis by nitric oxide synthase (NOS) willaid in drug development (for hypertension, atherosclerosis, diabetes) and therapeutic treatments (sickle cellanemia, blood substitutes, and septic shock) that utilize NO bioactivity. Determining catalytic and regulatorymechanisms of NOS is critical for understanding how NO is produced and managed physiologically, and fordesigning therapeutic agents that target NOS function. Determining the molecular mechanisms behind theregulation and physiological production of NO by NOS is our research goal. Our objective is determining howthe kinetics of CO, NO, and 62 binding to NOS are controlled by conformational changes induced bycofactors and substrate. Our hypothesis is that the binding of substrates and cofactors has a direct effect onthe reactivity and accessibility of the active site. Our rationale is that understanding the modulation of ligandbinding and heme reactivity by substrate and cofactor binding is crucial for under-standing how NO isproduced and managed endogenously. We will use a specialized multichannel (200-800 nm) laser-basednanosecond time-resolved spectrophotometer to measure the fast kinetics of ligand binding, electrontransfer,and oxygen activation involved in NO synthesis as a function of the binding of substrate andcofactors.
Our aims are: 1) By measuring CO bimolecular recombination kinetics as a function of cofactorinteractions, determine the structural mechanism for the binding of cofactors altering the reactivity of NOS.Our hypothesis is that the binding of cofactors modulates heme reactivity by inducing conformationalchanges. 2) Determine how NOS controls the binding and release of NO by measuring recombinationkinetics as a function of cofactor interactions. Our hypothesis is that binding cofactors causes structuralchanges, altering the binding kinetics of NO. 3) Determine the structural mechanism behind CaM regulationin neuronal NOS. The PI hypothesizes that control elements in the reductase domain affect the reactivity ofthe active site. 4) Determine how the binding of cofactors alters reactivity to oxygen and alters electrontransfer reactions of NOS. Our hypothesis is that 02 binding and kinetics are influenced by the binding ofcofactors. We will examine the kinetics of oxygen binding and the formation of oxygen activatedintermediates in neuronal NOS (nNOS) using nanosecond multichannel absorption spectroscopy after flowflashinitiation of the reaction with Oz- Relevance to Public Health: Knowledge of the specific molecularmechanisms of how NO is produced and managed physiologically by the binding of substrates and cofactorsis crucial to understanding and controlling NO physiology and understanding how compromised NOphysiology leads to deleterious health effects.
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