Abstract
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.
Highlights
nitric oxide (NO) is a product of the Nitric oxide synthase (NOS) reaction, but is believed to tightly bind to the heme iron [10, 12, 19]
Spectral studies of the NO-ferric hemoproteins or watersoluble ferric heme complexes have to date been extremely difficult because of their intrinsic instability toward autoreduction and of NO instability under aerobic conditions [20, 23, 24]
In order to understand the interaction between NO and the P450 heme active site, we studied the NO binding to the wildtype and putative distal mutant P450 1A2 enzymes by taking advantage of the NO donor
Summary
NO is a product of the NOS reaction, but is believed to tightly bind to the heme iron [10, 12, 19]. An imidazole-coordinated hemoprotein, metHb, is reduced by the binding of NO under anaerobic conditions [23]. The NO complex stability of the thiolate-coordinated ferric hemoproteins, appeared irreconcilable with the strong electron-donating capability of the cysteine thiolate. Spectral studies of the NO-ferric hemoproteins or watersoluble ferric heme complexes have to date been extremely difficult because of their intrinsic instability toward autoreduction and of NO instability under aerobic conditions [20, 23, 24]. Most of the NO binding studies to hemoproteins have been done for ferrous complexes under anaerobic conditions In order to understand the interaction between NO and the P450 heme active site, we studied the NO binding to the wildtype and putative distal mutant P450 1A2 enzymes by taking advantage of the NO donor. We discuss the Fe-N-O conformation in the P450 1A2 distal site in association with the NOS distal structure
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