Binding mode analysis of the NADH cofactor in nitric oxide reductase: a theoretical study

Nitric oxide reductase (Nor), a member of the cytochrome P450 superfamily, takes part in the denitrification process of fungi by reducing NO to N(2)O. Evidence indicates that Nor binds NADH, source of the reducing equivalents of the reaction, within its large hydrophilic ligand binding cavity on the distal side of heme and receives electrons directly from the cofactor. Here we present a binding mode analysis of the structure of the Nor-NO-NADH complex, performed in three steps. The NADH cofactor was first docked into the enzyme interior using the Monte Carlo multiple minimum algorithm, refined by low-mode conformational search and the final arrangement was obtained in a 5ns NPT molecular dynamics simulation. The NADH cofactor, in our results, is positioned--by Arg174, Lys291, Asp393 and several water molecules--within reactive distance of the NO binding spot suggesting a direct hydride shift mechanism between the two. The catalytically required water molecule is captured by NADH and the cofactor not only retains the suggested H-bonded proton transfer pathway between the active site and the solvent, but provides structural restraint for its members. We also found that direct interaction is formed between the cofactor and propionate A of the heme group, which flips from the proximal to the distal side of the heme plane in order to become an H-bonding partner of NADH. The role of Arg64 and Glu71 was suggested to be fixing the residues of the translocated helix B' to their new position.