Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical.
Nitric oxide (NO) is synthesized in mammals where it acts as a signal molecule for neurotransmission, vasorelaxation, and cytotoxicity. The NO synthases isolated from brain and cytokine-activated macrophages are FAD- and FMN-containing flavoproteins that display considerable sequence homology to NADPH-cytochrome P-450 reductase. However, the nature of their catalytic centers is unknown. We have found that both isoenzymes contain 2 mol of iron-protoporphyrin IX/mol of enzyme homodimer. The optical and EPR spectroscopic properties of the heme groups were found to be remarkably similar to those of high-spin cytochrome P-450. The heme iron in the resting NO synthase is ferric and five-coordinate with a cysteine thiolate as the proximal axial ligand. In addition, the EPR spectra of the resting NO synthases contained a free radical signal attributable to a bound flavin semiquinone that appeared to interact magnetically with the ferric heme iron. NO production was inhibited by carbon monoxide, implying a role for the heme groups in catalysis.
Stuehr
D J
DJ
Immunology Section, Cleveland Clinic, Ohio 44195.
Ikeda-Saito
M
M
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D019001:Nitric Oxide Synthase; D051381:Rats; D013053:Spectrophotometry; D014162:Transfection
D006420:Hemeproteins; D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; D003577:Cytochrome P-450 Enzyme System; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases