IRON-LIGAND STRUCTURE AND IRON REDOX PROPERTY OF NITRIC-OXIDE REDUCTASE CYTOCHROME P450NOR FROM FUSARIUM-OXYSPORUM - RELEVANCE TO ITS NO REDUCTION ACTIVITY

We studied the nitric oxide reductase, cytochrome P450nor, purified from a denitrifying fungus Fusarium oxysporum with electron paramagnetic resonance spectral and redox potential measurements, The EPR spectral features of P450nor in the ferric resting, the ferric cyanide-bound, and the ferrous NO-bound forms were the same as the corresponding ones of other general P450s such as Pseudomonas putida P450cam. In contrast, the metyrapone complex of ferric P450nor gave an EPR spectrum with significantly different g values from that of P450cam. The EPR results were explained in terms of similarity in the immediate configuration of the S--Fe-ligand (H2O, CN-, NO) structure between P450nor and P450cam but a structural difference at the heme distal pocket, especially in the substrate binding domain; P450cam has a camphor binding domain, while P450nor does not, In spite of the same S--Fe-H2O configuration, the redox potential of P450nor in the ferric/ferrous couple was measured to be -307 mV, which is much lower than those of the camphor-bound (-140 mV) and -free (-250 mV) P450cam. The lower redox potential could be attributable to the different electrostatic interaction of the heme with its surroundings; e.g., the heme environment of P450nor is charged either more negatively or less positively than P450cam. The low redox potential of P450nor is favorable for the reductive activation of the NO Ligand, in good consistency with its reaction mechanism which we have proposed [Shire et al, (1995) J. Biol, Chem. (in press)], where the NO Ligand bound to the ferric iron is reduced with two electrons from NADH and then reacts with another NO, yielding N2O and H2O. The EPR and redox data were also discussed in relation to the unique properties of P450nor.