TCB Publications - Abstract

TCB Publications - Abstract

Paween Mahinthichaichan, Robert B. Gennis, and Emad Tajkhorshid. Bacterial denitrifying nitric oxide reductases and aerobic respiratory terminal oxidases use similar delivery pathways for their molecular substrates. Biochimica et Biophysica Acta - Bioenergetics, 1859:712-724, 2018. (PMC: PMC6078787)

MAHI2018B-ET The superfamily of heme-copper oxidoreductases (HCOs) include both NO and O$_2$ reductases. Nitric oxide reductases (NORs) are bacterial membrane enzymes that catalyze an intermediate step of denitrification by reducing nitric oxide (NO) to nitrous oxide (N$_{2}$O). They are structurally similar to heme-copper oxygen reductases (HCOs), which reduce O$_{2}$ to water. The experimentally observed apparent bimolecular rate constant of NO delivery to the deeply buried catalytic site of NORs was previously reported to approach the diffusion-controlled limit (10$^{8}$-10$^{9}$ M$^{-1}$s$^{-1}$). Using the crystal structure of cytochrome-c dependent NOR (cNOR) from Pseudomonas aeruginosa, we employed several protocols of molecular dynamics (MD) simulation, which include flooding simulations of NO molecules, implicit ligand sampling and umbrella sampling simulations, to elucidate how NO in solution accesses the catalytic site of this cNOR. The results show that NO partitions into the membrane, enters the enzyme from the lipid bilayer and diffuses to the catalytic site via a hydrophobic tunnel that is resolved in the crystal structures. This is similar to what has been found for O$_2$ diffusion through the closely related O$_2$ reductases. The apparent second order rate constant approximated using the simulation data is $\sim$5$\times$$10^{8}$M$^{-1}$s$^{-1}$, which is optimized by the dynamics of the amino acid side chains lining in the tunnel. It is concluded that both NO and O$_2$ reductases utilize well defined hydrophobic tunnels to assure that substrate diffusion to the buried catalytic sites is not rate limiting under physiological conditions.

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