Conserved Glu318 at the cytochrome P450 1A2 distal site is crucial in the nitric oxide complex stability.
Nitric oxide synthase (NOS) has a thiolate-coordinated heme active site similar to that of cytochrome P450 (P450). Both NOS and P450 form stable nitric oxide (NO)-ferric heme complexes, whereas an NO-ferric heme complex of methemoglobin, that has an imidazole-coordinated heme active site, is easily reduced. The NO complex stability of the thiolate-coordinated hemoproteins, however, appeared irreconcilable with the strong electron-donating capability of the cysteine thiolate. In the present study, NO bindings to cytochrome P450 1A2 (P450 1A2) distal mutants were studied in the presence of various substrates. We found that a mutation at Glu-318 to Ala in the putative distal site of P450 1A2, suggested to be important in the O2 activation of P450 reactions, markedly facilitates the reduction of the NO-ferric complex. Addition of 1,2:3,4-dibenzanthracene or phenanthrene almost abolished the mutation effect on the NO complex. Based on these results, together with other spectral and kinetic data, it is suggested that the NO-ferric complex stability of P450, and perhaps of NOS, is largely ascribed to an ionic bridge between NO and the distal carboxyl group.
Nakano
R
R
Institute for Chemical Reaction Science, Tohoku University, Sendai 980-77, Japan.
Sato
H
H
Watanabe
A
A
Ito
O
O
Shimizu
T
T
D001665:Binding Sites; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D005290:Ferric Compounds; D005971:Glutamates / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D011994:Recombinant Proteins; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D005290:Ferric Compounds; D005971:Glutamates; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2