Nitric oxide synthase (NOS) produces nitric oxide (NO) and citrulline from arginine, molecular oxygen, and NADPH. NO plays a prominent role in mammals as a mechanism of cytotoxicity for macrophages , and as a signaling molecule involved in neurotransmission, in regulation of blood flow in the vascular system, and in the perfusion and function of many organs and tissues. Mammalian NOS exists in three isoforms (inducible, endothelial, and neuronal) and is comprised of an N-terminal oxygenase domain containing cysteine-ligated heme and tetrahydrobiopterin (H4B) cofactors, a C-terminal reductase domain that binds flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), and an intervening calmodulin binding region . The chemistry of NOS occurs in two mechanistically distinct steps with N-hydroxyarginine (NHA) as a stable intermediate. In both steps, oxygen binds at heme and is activated by electron transfer from the reductase domain and the proximal H4B. Electron transfer from the pterin cofactor to the heme is the rate limiting step for both Arg and NHA oxidation.

We have succeeded in working out methods to express both the inducible isoform of NOS (iNOS) and the neuronal isoform (nNOS), generated unnatural substrate analogues and inhibitors, and developed methods for incorporation of unnatural amino acids, pterin, and heme cofactors. This work is aimed at understanding the mechanism of NO biosynthesis on a molecular level. Current projects are focused on the role of the pterin cofactor (H4B) in NOS catalysis in both Arg and NHA oxidation, as well as the fundamental chemical steps and intermediates involved in substrate oxidation. Pterin is believed to serve as a redox active cofactor and donates an electron to heme in the activation of molecular oxygen at heme. The level of oxygen activation is believed to be substrate dependent, and both high valent, Fe(IV)=O(por•+) and Fe(III)-OOH species, termed Compound I and 0, respectively) are implicated in catalysis. How the protein active site directs formation and reactivity of these highly oxidizing species is a topic of intense research.

More recently, many species of bacteria have been identified with NOS-like enzymes in their genome . Most of these so-called bacterial NOS enzymes are comprised of a single domain with high sequence homology to the oxygenase domain of mammalian NOS. Many of these bacteria do not contain the genes encoding for the machinery for H4B production. These bacteria do, on the other hand, contain the genes for tetrahydrofolate (H4F) production, which contains the pteridine ring system responsible for the redox function of H4B in mammalian NOS. We are generally interested in these new NOS-like systems. Do these bacterial NOS-like proteins produce NO? What cofactors are required for their activity? How do the mechanisms of NO synthesis compare and contrast to the mammalian systems?

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