Conserved Glu at the Cytochrome P450 1A2 Distal Site Is Crucial in the Nitric Oxide Complex Stability

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 O 2 activation of P450 reactions, markedly facilitates the reduction of the NOferric 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. The microsomal P4501 class of hemoproteins catalyze monooxidation reactions of a variety of external and endogenous organic compounds with O 2 and electrons (Refs. 1-3 and references therein). Likewise, NOS produces physiologically important NO from L-Arg and N G -hydroxy-L-Arg in the presence of O 2 and NADPH (Refs. 4 -7 and references therein). A hemoprotein domain of NOS has a monooxidation catalytic site that is likely to have a thiolate-coordinated heme iron similar to that of P450 (8 -18).NO is a product of the NOS reaction, but is believed to tightly bind to the heme iron (10,12,19). The NO-ferric NOS complex is stable in the absence of O 2 , and is similar to P450 (10, 12, 19 -22). On the other hand, an imidazole-coordinated hemoprotein, metHb, is easily reduced by the binding of NO under anaerobic conditions (23). NO-ferric-porphyrin complexes are also easily reduced in polar solvents (24). The NO complex stability of the thiolate-coordinated ferric hemoproteins, thus, 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). Thus, most of the NO binding studies to hemoproteins have been done for ferrous complexes under anaerobic conditions (Refs. 25-29 and references therein). Recently, it became feasible to carry out NO-binding studies for ferric hemoproteins as well as ferrous hemoproteins, because of the current commercial availability of NO-releasing (donating) reagents such as NOR1. With the use of this NO donor, spectral titration experiments of NO and laser flash photolysis experiments for ferric P450 enzyme are possible.In order to understand the interaction between NO and t...

The Heme-regulated Eukaryotic Initiation Factor 2α Kinase

“…The ability of CO to antagonize NO-induced activation of HRI leads us to propose a novel feedback mechanism through which protein synthesis may be regulated by these diffusible gases. Increases in cellular NO concentrations cause the release of proteinbound heme (71)(72)(73), and the induction of HO-1 (11). The HO-1-catalyzed breakdown of heme would not only be critical in protecting cells from oxidative damage that would result from the presence of elevated concentrations of free heme, but would also produce elevated concentrations of CO, which could act as a feedback inhibitor of HRI activation induced by NO.…”

Binding of Oxygen and Carbon Monoxide to a Heme-regulated Phosphodiesterase from Escherichia coli

“…Anaerobic spectral experiments were conducted on a Shimadzu UV-160A spectrophotometer in a glove box under a nitrogen atmosphere with an O 2 concentration of less than 50 ppm (22)(23)(24). Following reduction of the heme by sodium dithionite, excess dithionite was removed using a Sephadex G-25 column in the glove box.…”

Crucial Role of Lys423 in the Electron Transfer of Neuronal Nitric-oxide Synthase

“…nNOS was expressed in Saccharomyces cerevisiae using the acid phosphatase promoter previously used for the expression of cytochrome P450 1A2 (25)(26)(27). The oligonucleotide primers for the mutations of Lys 423 to Glu, Met, Leu, and Asn were 5Ј-CCAGTGGTCCGAGCTGCA-GG-3Ј, 5Ј-CAGTGGTCCATGCTGCAGGT-3Ј, 5Ј-CAGTGGTCCCTGCT-GCAGGT-3Ј, and 5Ј-AGTGGTCCAATCTGCAGGTG-3Ј, respectively.…”