Simulation study of interactions and reactivities between NADH cytochrome b5 reductase and cytochrome b5

The purpose of this study is to investigate the structures and reactivities for the docking complex of cytochrome b5 (b5) and NADH-cytochrome b5 reductase (b5R). The authors performed a molecular dynamics (MD) simulation and quantum mechanical/molecular mechanical (QM/MM) calculations of our docking model. The binding free energy analyses for a 2 ns MD trajectory revealed the van der Waals interaction and non-polar contribution of the solvation played important roles in the inter-protein interaction in water. These non-electrostatic interactions came from the relatively large contact area between b5 and b5R proteins (ca. 1370 Å2). Computational alanine scanning analysis of 78 residues around the b5-b5R interface has been done. These analyses indicate that both Lys162 and Lys163 in b5R are important residues in the binding free energy, which are coincident with results from experimental steady state enzymatic activities. Our results suggest that Arg91, Arg142, and Lys294 in b5R also participate in the interaction with b5, whereas no significant effect is obtained by mutagenesis of b5 except for Thr65Ala mutant. Based on our reaction mechanism of H− transfer from NADH to FAD in the vacuum state, the charge embedded QM/MM calculations have been applied to the hydrated b5-b5R docking structure. Most important conclusion provided by our calculations is that the product becomes more stable than the reactant by the presence of heme (Fe3+). The activation energy in the b5-b5R complex is also smaller than that without heme.