Heme coordination and structure of the catalytic site in nitric oxide synthase.
Nitric oxide (NO), recently found to play many physiological roles, is generated by the catalysis of L-arginine and O2 to L-citrulline and NO by nitric oxide synthases (NOSs). Resonance Raman spectra from the heme of resting, reduced, and CO-bound forms of rat brain NOS firmly establish that the enzyme belongs to the P-450 class of enzymes. The electron density marker line (V4) in the Raman spectrum of ligand-free ferrous NOS has a low frequency (1347 cm-1), indicating a thiolate axial ligand on the heme. The assignment of a thiolate axial ligand is confirmed in the CO-bound form of the enzyme by the frequency of the Fe-C-O bending mode at 562 cm-1. The heme is resting NOS is five-coordinate high spin and thereby differs from the resting state of most substrate-free P-450s, which are predominantly six-coordinate low spin. The frequency of the Fe-CO stretching mode in the CO-bound enzyme at 491 cm-1, identified by isotope substitution, in higher than that in substrate-free P-450s. Thus, in the ferric and the CO-bound forms of the enzyme, the sixth-ligand binding site on the heme is restricted by steric or hydrophobic interactions. In addition, the Fe-CO stretching mode is broad (30 cm-1) and may be resolved into two overlapping lines of equal intensity, indicating that the heme domains can adopt two distinct conformations.
Wang
J
J
AT&T Bell Laboratories, Murray Hill, New Jersey 07974.
Stuehr
D J
DJ
Ikeda-Saito
M
M
Rousseau
D L
DL
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002248:Carbon Monoxide / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D007668:Kidney; D019001:Nitric Oxide Synthase; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D014162:Transfection
D011994:Recombinant Proteins; D006418:Heme; D002248:Carbon Monoxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases