Characterizations of Substrate Delivery Pathways in the Nitric Oxide Reductase

Abstract

Nitric oxide reductase (NOR), a structurally homologous enzyme of the heme-copper oxidoreductase superfamily and an integral membrane protein, carries out the intermediate step of denitrification by reducing nitric oxide (NO) to nitrous oxide (N2O). The reduction reaction occurs at the buried heme-iron complex. However, experiments demonstrated that the NO binds NOR at a rate near the diffusion limit (5×108 M−1s−1), and that NOR requires only a few nanomolar of NO to reach its half-maximal reduction activity. To provide atomistic and quantitative descriptions of the NO delivery mechanism, molecular dynamics (MD) simulations have been performed, using the crystal structure of cytochrome-c dependent NOR (cNOR) from Pseumonas aeruginosa as the starting model. Independent explicit ligand sampling simulations with high NO concentration have been carried out to accelerate the sampling of NO within the simulation timescale. The results indicate that substrate NO enters the enzyme via three membrane-accessible hydrophobic tunnels, two of which correspond to the observed xenon-bound hydrophobic tunnel of B-type HCO from Thermus thermophilus (cytochrome ba3) by X-ray crystallography. All three tunnels merge into one pathway. Two bottlenecks of NO migration, composed of highly conserved residues, are identified. One is at residues I65 and W209, located 15A from the reduction site, while the other is at residue V206, located 6-8A from the reduction site. Umbrella sampling simulations and in silico mutations have also been performed to characterize the free energy of NO insertion along the pathway and confirm the identified bottlenecks. Alanine substitutions of the bottleneck residues result a decrease in the free-energy barriers, and result in an increase of NO delivery events. The study demonstrates that pathways facilitate the delivery of small gases, such as NO, in enzymes.