Radu Silaghi-Dumitrescu

NITRIC OXIDE REDUCTION BY P450NOR

P450NOR is a heme enzyme employed by fungi to reduce nitric oxide, a toxic intermediate which is produced in these organisms upon nitrate reduction. The electrons required for this reduction are provided by NADH:

Known steps of the P450nor reaction, from experiment:

We have engaged in computational investigations of the P450NOR, employing density functional calculations (DFT) and molecular mechanics-based docking. Emphasis has been placed on establishing the structure of the experimentally-observed ‘444-nm intermediate’; candidates for this species have included, among others, such structures as [FeNO]⁷, [FeNO]⁸, [FeNOH]⁸, [FeN(H)O]⁸, or [FeN(H)OH]⁸. The issue of outer-sphere vs. inner-sphere electron transfer has also been examined.

NADH docking onto the P450nor ferric-NO adduct. A: View perpendicular to the solvent-exposed heme. B: side-view, along the NADH-binding crevice. C: close-up of the active side. Protein backbone-blue, heme-red, NO-green, NADH-yellow.

• A one-electron mechanism, involving [FeNO]⁷ as intermediate, is disfavored by the strong endothermicity of the [FeNO]⁷ ® [FeNO]⁸ step, and by the fact that [FeNOH]⁸ is less stable than its isomer [FeN(H)O]⁸

• A hydride (two-electron) mechanism (initially proposed by Averill) is favored by the above arguments and by experimentally-observed NADH/NADD kinetic isotope effects

• The docking results suggest that the oxygen atom in the [FeNO]⁶ complex of P450nor is in a better position than the nitrogen atom to receive the hydride from NADH.

• The 444-nm intermediate is proposed to be [FeNOH]⁸ (or, cf. subsequent results from other computational groups, the diprotonated version, [FeN(H)OH]⁸)

• The NADH-binding crevice identified by the docking results contains two residues previously proven by site-directed mutagenesis to be involved in NADH binding