Bacterial denitrifying nitric oxide reductases and aerobic respiratory terminal oxidases use similar delivery pathways for their molecular substrates

Abstract

The superfamily of heme‑copper oxidoreductases (HCOs) include both NO and O reductases. Nitric oxide reductases (NORs) are bacterial membrane enzymes that catalyze an intermediate step of denitrification by reducing nitric oxide (NO) to nitrous oxide (NO). They are structurally similar to heme‑copper oxygen reductases (HCOs), which reduce O 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 (10-10 M s). 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 diffusion through the closely related O reductases. The apparent second order rate constant approximated using the simulation data is ~5 × 10 M s, which is optimized by the dynamics of the amino acid side chains lining in the tunnel. It is concluded that both NO and O reductases utilize well defined hydrophobic tunnels to assure that substrate diffusion to the buried catalytic sites is not rate limiting under physiological conditions.

Selective Permeability of Carboxysome Shell Pores to Anionic Molecules

“…The movement of a molecule through a protein may involve not only structural perturbations of the protein, from breathing motions of lining amino acids 49–52 to largescale conformational changes, 53–57 but also the dynamics of the passing molecule (substrate or ligand). 58 Although the pores of CsoS1A and CcmK4 are relatively wide to accommodate a free passage of small molecules, such as HCO3−, CO 2 and O 2 , the calculated ΔG profiles indicated that such molecules cannot pass through easily.…”

Mimochrome, a metalloporphyrin‐based catalytic Swiss knife†

“…While NORs use exclusively heme b and bind iron (Fe B ) in their nonheme center with a conserved 3His-1Glu first coordination sphere, HCOs may host different types of hemes (e.g., heme o, a, and b) and bind copper (Cu B ) with a tripodal 3His coordination sphere, depending on the organism [73]. Thorough mechanistic studies have been performed for these two homologous classes of proteins [74][75][76][77][78][79] and few details are still under debate [80][81][82]. Nonetheless, in both cases protein matrix is able to promote selectivity of one metal over the other and to drive the correct inter-metal distance along the various oxidation states of the metal and reaction intermediates [83].…”

Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo–electron microscopy

“…S6), it may compensate for the loss of Val 485 to continue shuttling NO. A computation study on Pa cNOR investigated NO diffusion and found that several migration pathways exist (termed dominant and alternate), where migration of NO is ~10-fold lower using the alternate route than using the dominant pathway ( 49 ). Several of the routes include conserved residues found in our tunnel analysis (Val 285 , Ile 484 , Leu 336 , and Leu 487 ), which may suggest a generic role in NO transport across NORs.…”