Nitric oxide (NO), a recently discovered mammalian metabolite, is important in neural function, maintenance of smooth muscle tone, and immune response. It is formed oxidatively from arginine by the enzyme nitric oxide synthase (NOS). The first step, the hydroxylation of arginine to N-hydroxyarginine, is followed by the three-electron oxidation to give products, citrulline and NO. NOS is an oxygen- and NADPH dependent flavoprotein with a P450-type heme and reduced biopterin. Incorporation of metalloporphyrins instead of the natural one, iron protoporphyrin IX, is possible using a baculovirus expression system. These metal-substituted derivatives (Zn, Co, Mn) will be used to investigate the mechanism of oxidation of arginine. Optical spectroscopy, electron paramagnetic resonance, and circular dichroism will be used to characterize the structural and electronic features of these derivatives in the presence and absence of substrates, inhibitors and ligands. Electron transfer will be assayed by NADPH oxidation and cytochrome c reduction. The hydroxylation of arginine may occur at the heme or at reduced biopterin, the presence of which is required for catalysis. The cobalt- and manganese-substituted enzymes will be useful for examining the mechanism of the first hydroxylation step if pterin is not involved. The second oxidative step occurs at the heme. The derivatives will be studied for their ability to catalyze the oxidation of N-hydroxyarginine with )O2/NADPH and with peroxides and oxygen-atom donors. These mechanistic studies will enhance our understanding of this important enzyme and its ubiquitous co-factors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM017944-03
Application #
2518831
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1997-09-01
Project End
Budget Start
1997-09-01
Budget End
1998-02-28
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Pharmacy
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Richards, M K; Clague, M J; Marletta, M A (1996) Characterization of C415 mutants of neuronal nitric oxide synthase. Biochemistry 35:7772-80