Tetrahydrobiopterin-deficient nitric oxide synthase has a modified heme environment and forms a cytochrome P-420 analogue.
Optical absorption and resonance Raman spectra of neuronal nitric oxide synthase (b-NOS) isolated in the absence of tetrahydrobiopterin demonstrate that the enzyme preparation is very unstable. This unstable form of the enzyme has properties analogous to those of cytochrome P-420cam, an inactive form of cytochrome P-450cam. Although cysteine is preserved as the proximal ligand in both the ferric and ferrous forms of unstable b-NOS, the lack of tetrahydrobiopterin significantly increases the hexacoordinate low-spin fraction of the heme content, resulting in a loss of the enzymatic activity. Upon the addition of CO, the unstable b-NOS converts from a species exhibiting a Soret absorption maximum at 443 nm, as reported for the CO adducts of stable b-NOS and cytochrome P-450cam, to a species with a Soret maximum at 421 nm. The resonance Raman spectrum of the 421-nm form is the same as those of CO-bound myoglobin at low pH and CO-bound cytochrome P-420cam. The heme in this form of the enzyme is coordinated by a weaker ligand than thiolate; histidine coordination in the CO-bound form of the P-420-like species of NOS is consistent with all of the available data. A similar unstable form of the macrophage (i-NOS) enzyme was also detected. Not only does the heme pocket of NOS have the same coordination as cytochrome P-450 in its stable form, but the partially denatured form has the same properties as cytochrome P-420, the inactive form of cytochrome P-450. Among other possible roles, tetrahydrobiopterin may play a significant role in the stabilization of the active enzyme.
Wang
J
J
AT&T Bell Laboratories, Murray Hill, New Jersey 07974, USA.
Stuehr
D J
DJ
Rousseau
D L
DL
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D002460:Cell Line; D003580:Cytochromes / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D051379:Mice; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase; D013059:Spectrum Analysis, Raman
D003580:Cytochromes; D001708:Biopterins; D006418:Heme; C013464:cytochrome P420; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C003402:sapropterin