Identification of nitric oxide synthase as a thiolate-ligated heme protein using magnetic circular dichroism spectroscopy. Comparison with cytochrome P-450-CAM and chloroperoxidase.
Nitric oxide (NO) has recently been recognized as an important biomolecule playing diverse physiological roles. It is synthesized in several different tissues from L-Arg and O2, using NADPH as an electron donor, by a family of heme-containing catalytically self-sufficient monooxygenases known as nitric oxide synthases (NOS). Recently, the CO complex of reduced NOS has been shown to exhibit an absorption maximum near 450 nm, a characteristic spectral feature of cytochrome P-450 (P-450). Yet, the amino acid sequences of NOS and P-450 have no homology. To further probe the active site heme coordination structure and the heme environment of NOS, we have employed magnetic circular dichroism (MCD) and CD spectroscopy in the present study. MCD spectra of several derivatives of rat brain neuronal NOS strikingly resemble those of analogous derivatives of bacterial P-450-CAM and fungal chloroperoxidase, two known thiolate-ligated heme proteins. Given the proven fingerprinting capability of MCD spectroscopy, this provides convincing evidence for endogenous thiolate (cysteinate) ligation to the heme iron of NOS. Furthermore, the heme-related Soret CD bands of NOS (positive) and P-450s (negative), as represented by P-450-CAM, are almost mirror images, whereas chloroperoxidase exhibits totally different CD band shapes. This suggests that the active sites of NOS and P-450 may share some common structural features, but significant distinctions exist between their heme environments in certain aspects such as hydrophobicity or size.
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Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA.
Stuehr
D J
DJ
Ikeda-Saito
M
M
Dawson
J H
JH
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D019475:Camphor 5-Monooxygenase; D002478:Cells, Cultured; D002711:Chloride Peroxidase / Q000737:chemistry*; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D015394:Molecular Structure; D019001:Nitric Oxide Synthase; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
D006420:Hemeproteins; D011994:Recombinant Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D002711:Chloride Peroxidase; D019001:Nitric Oxide Synthase; D019475:Camphor 5-Monooxygenase; D000594:Amino Acid Oxidoreductases