Crystal structures of cytochrome P450nor and its mutants (Ser286 -> Val, Thr) in the ferric resting state at cryogenic temperature: a comparative analysis with monooxygenase cytochrome P450s

Cytochrome P450nor (P450nor) is a heme enzyme isolated from the denitrifyin g fungus Fusarium oxysporum and catalyzes the NO reduction to N2O. Crystal structures of the wild type and two Ser286 mutants (Ser286-->Val, Ser286--> Thr) of P450nor have been determined for the ferric resting forms at a 1.7 Angstrom resolution at cryogenic temperature (100 K). We carried out three comparative analyses: (1) between the structures of P450nor at room tempera ture and cryogenic temperature, (2) between the structures of P450nor and f our monooxygenase P450s, and (3) between the structures of the WT and the S er286 mutant enzymes of P450nor. Comparison of the charge distribution on t he protein surface suggests that proton and electron flow to the heme site is quite different in P450nor than in monooxygenase P450s. On the basis of the mutant structures, it was found that a special hydrogen-bonding network , Wat99-Ser286-Wat39-Asp393-solvent, acts as a proton delivery pathway in N O reduction by P450nor. In addition, the positively charged cluster located beneath the B'-helix is suggested as possible NADH binding site in P450nor , from which the direct two-electron transfer to the heme site allows to ge nerate the characteristic intermediate in the NO reduction. These structura l characteristics were not observed in structures of monooxygenase P450s, i mplying that these are factors determining the unique NO reduction activity of P450nor. (C) 2000 Elsevier Science S.A. All rights reserved.