Nitric oxide synthase mechanisms

Abstract

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from molecular oxygen and L-arginine via a consecutive two-step reaction by using NADPH as the electron source. In the first step, L-Arginine is hydroxylated to N-hydroxyarginine (NOHA). In the second step, NOHA is oxidized to citrulline and NO.;The mechanisms underlying the functionality of NOS, including the role of L-Arginine and H4B in the enzyme's reaction cycle and self-regulation, are not well-understood. In order to better our understanding of these mechanisms, we have conducted numerous spectroscopic studies on the iNOSoxy isoform. Optical absorption and resonance Raman spectroscopies served as the fundamental techniques used in our studies of iNOSoxy. In addition, electron paramagnetic resonance and mass spectroscopies were performed in order to identify changes in the protein matrix surrounding the heme environment. In the course of our studies, we have studied the iNOSoxy enzyme in a variety of enzymatic conditions, including transient reaction intermediates. In the cases where transient intermediates were studied, rapid sub-millisecond mixing was coupled to resonance Raman spectroscopy.;From studies conducted on the carbon monoxide, NO, and cyanide bound adducts of iNOSoxy (Chapter 2), we discovered that the differential interactions between the various heme ligands and the protein matrix in response to L-Arg and/or H4B binding is coupled to heme distortions. Further studies conducted in the absence of H4B demonstrate NO induced heme active site changes that are dependent on the monomer/dimer equilibrium of iNOS (Chapter 3). In Chapter 5, we demonstrate the H4B dependent reduction of the ferric-NO bound enzyme in the presence of NOHA. In Chapter 4, the influence of L-Arg and H4B analogues on the ferric-NO bound enzyme was investigated in order to gain further insights into the modulation of the autoinhibitory complex. In a final study (Chapter 6), the role of substrate in the NOS reaction cycle was explored through the investigation of its influence on NOS oxygen intermediates. Overall, our studies reveal diverse interactions between L-Arg, H4B, NOHA, their respective analogues, and the iNOS isoform that gives multiple insights into the mechanisms that govern NOS functionality.