Cytochrome P450 purified from Fusarium oxysporum (P450nor) is a unique heme enzyme that catalyzes the reduction of nitric oxide to nitrous oxide with electrons directly transferred from NADH (2NO + NADH + H+--> N2O + H2O + NAD+). We studied the reaction of P450nor with NO and NADH using stopped-flow rapid scan and low temperature spectroscopic methods. The NO ligand can bind to the ferric enzyme to form the stable NO bound complex, P450nor(Fe3+NO). Reduction of P450nor(Fe3+NO) with NADH yielded an intermediate, which transiently formed (tau = approximately 100 ms) and spontaneously decomposed to the Fe3+ state. The optical absorption spectrum of the intermediate was different from that of P450nor(Fe2+NO), which was formed by either a one-electron reduction of P450nor(Fe3+NO) with Na2S2O4 or NO binding to P450nor(Fe2+). On the basis of these observations, we suggested that the intermediate is presumably a two-electron reduced product of P450nor(Fe3+NO) by NADH, formally the (Fe3+NO)2-complex. We determined the rate constants of these reactions at 10 degrees C for the NO binding to P450nor(Fe3+) (2.6 x 10(7) M-1 s-1), the NADH reduction of P450nor(Fe3+NO) (0.9 x 10(6) M-1 s-1), and the spontaneous decomposition of the intermediate (0.027 s-1). In these kinetic measurements, it was found that the former two processes are fast enough, while the latter is extremely slow, compared with the fast turnover of the catalytic reaction (1200 s-1 at 10 degrees C), which we measured by monitoring the NADH consumption. Therefore, we suggested that in the catalytic cycle, decomposition of the intermediate is fairly accelerated by free NO, resulting in such a fast turnover. On the basis of several lines of the spectroscopic and the kinetic evidence, we proposed a possible mechanism of the NO reduction by P450nor.

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 A resolution. These structures provide significant new insights into how NO is reduced in biological systems. The haem distal pocket is open to solvent, implicating this region as a possible NADH binding site. In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

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Nitric oxide reductase from the denitrifying fungus Fusarium oxysporum catalyzes the reduction of NO to N 2 O [Nakahara, K., et al. J. Biol. Chem. 1993, 268, 8350-8355]. Since this enzyme belongs to the cytochrome P450 superfamily [Kizawa, H., et al. J. Biol. Chem. 1991, 266, 10632-10637], it is called cytochrome P450nor (P450nor), but does not exhibit monooxygenation activity. In the present study, we examine the coordination structure of the heme iron in P450nor in the ferric-NO form by using infrared, resonance Raman, and X-ray absorption (EXAFS ) extended X-ray absorption fine structure) spectroscopies, since the ferric-NO complex is a first intermediate in the NO reduction cycle by P450nor [Shiro, Y, et al J. Biol. Chem. 1995, 270, 1617-1623. We compared the data obtained with those for the d-camphor-bound form of Pseudomonas putida camphor hydroxylase cytochrome P450cam (P450cam), a typical model of the monooxygenase. From the vibrational spectroscopic measurements, we found that the Fe-bound N-O stretching frequency (ν(N-O)) occurred at 1851 cm -1 and the Fe-NO stretching frequency (ν(Fe-NO)) at 530 cm -1 for P450nor, while those at 1806 and 522 cm -1 were observed for P450cam, respectively. The assignments were confirmed by the 15 NO substituting effect on these vibrational frequencies. These results indicated that NO binds to the ferric iron in P450nor stronger than in P450cam. Support for this was provided from the EXAFS study, which gave an Fe-N NO bond distance of 1.66 ( 0.02 Å for P450nor and 1.76 ( 0.02 Å for P450cam. These spectroscopic results suggest that, compared with P450cam, the lower steric hindrance and/or the difference in the electrostatic interactions of the ligand NO with its surroundings facilitates the donation of the NO 2pπ* electron to the iron 3dπ orbital, resulting in the strengthening of the Fe-NO and the N-O bonds of P450nor. The vibrational spectral observation of the ferrous-CO complex of P450nor supported this suggestion. This configuration can reduce the electron density on the NO ligand in P450nor, which is seemingly relevant to the NO reduction reactivity of P450nor.

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The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of sixcoordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co 2؉ -substituted enzyme exhibited EPR signals of a broad g Ќ Ќ component and a g ሻ ሻ component with a poorly resolved triplet of 14 N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of ␣-subunits of Co 2؉ -hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co 2؉ -enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N 3 ؊ elicited the activation of the ferric enzyme by yielding five-coordinate high spin N 3 ؊ heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N 3 ؊ binding.

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