Spectroscopic and Kinetic Studies on Reaction of Cytochrome P450nor with Nitric Oxide

Abstract

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.

Involvement of a Glu71–Arg64 Couple in the Access Channel for NADH in Cytochrome P450nor

“…4) P450nor is the sole P450 that can receive electrons directly from NAD(P)H (NADH or NADPH) without the aid of a flavoprotein reductase or of iron-sulfur proteins. 4,5) The overall structure of P450nor determined by X-ray crystallography is similar to that of other P450s, whereas its unique features are also noticeable. 6) The B 0 -helix and F, G-loop that form the entrance of the heme-distal pocket extend outside to make a large cavity in the pocket.…”

“…A Rapidscan analysis was carried out as reported with a Unisoku Rapid Scan analyzer. 5) Absorption spectra of the E71A mutant in its resting ferric (Fig. 1) and reduced carbon monoxide-bound (data not shown) forms were identical to those of wild type P450nor, showing that the mutant protein had been adequately folded in the heterologous E. coli cells.…”

“…2, inset), an isosbestic point between the spectra of I and the resting ferric form. 5) In case of the mutant (Fig. 2B), however, accumulation of I was insufficient, and the ferric form (413 nm species) appeared in the late stage of the reaction.…”

“…The wild type (WT) or E71A mutant of P450nor (5 M final concentration) was mixed with NADH (20 M final) in a 50 mM TES buffer (pH 7.2) at 8 C, and each spectrum was recorded at indicated reaction time (the dead time of the apparatus is not included) with a Unisoku rapid-scan analyzer as reported. 5,7) The inset shows the time-dependent change in absorbance at 427 nm (A 427 ) that gave k obs . low spin state exchange in heme) in wild type P450nor (Fig.…”

“…1) form. 5) Thus, the k obs (apparent reduction rate) in the Iformation step can be obtained by tracing the timedependent change in absorbance at 427 nm (Fig. 2, inset), an isosbestic point between the spectra of I and the resting ferric form.…”