Fast Kinetic Investigations of Nitric Oxide Synthase

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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) will aid in drug development (for hypertension, atherosclerosis, diabetes) and therapeutic treatments (sickle cell anemia, blood substitutes, and septic shock) that utilize NO bioactivity. Determining catalytic and regulatory mechanisms of NOS is critical for understanding how NO is produced and managed physiologically, and for designing therapeutic agents that target NOS function. Determining the molecular mechanisms behind the regulation and physiological production of NO by NOS is our research goal. Our objective is determining how the kinetics of CO, NO, and 62 binding to NOS are controlled by conformational changes induced by cofactors and substrate. Our hypothesis is that the binding of substrates and cofactors has a direct effect on the reactivity and accessibility of the active site. Our rationale is that understanding the modulation of ligand binding and heme reactivity by substrate and cofactor binding is crucial for under-standing how NO is produced and managed endogenously. We will use a specialized multichannel (200-800 nm) laser-based nanosecond 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 and cofactors. Our aims are: 1) By measuring CO bimolecular recombination kinetics as a function of cofactor interactions, 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 conformational changes. 2) Determine how NOS controls the binding and release of NO by measuring recombination kinetics as a function of cofactor interactions. Our hypothesis is that binding cofactors causes structural changes, altering the binding kinetics of NO. 3) Determine the structural mechanism behind CaM regulation in neuronal NOS. The PI hypothesizes that control elements in the reductase domain affect the reactivity of the active site. 4) Determine how the binding of cofactors alters reactivity to oxygen and alters electron transfer reactions of NOS. Our hypothesis is that 02 binding and kinetics are influenced by the binding of cofactors. We will examine the kinetics of oxygen binding and the formation of oxygen activated intermediates in neuronal NOS (nNOS) using nanosecond multichannel absorption spectroscopy after flowflash initiation of the reaction with Oz- Relevance to Public Health: Knowledge of the specific molecular mechanisms of how NO is produced and managed physiologically by the binding of substrates and cofactors is crucial to understanding and controlling NO physiology and understanding how compromised NO physiology leads to deleterious health effects.