Nitric oxide (NO) plays diverse and significant roles in biological processes despite its cytotoxicity, raising the question of how biological systems control the action of NO to minimize its cytotoxicity in cells. As a great example of such a system, we found a possibility that NO-generating nitrite reductase (NiR) forms a complex with NOdecomposing membrane-integrated NO reductase (NOR) to efficiently capture NO immediately after its production by NiR in anaerobic nitrate respiration called denitrification. The 3.2-Å resolution structure of the complex of one NiR functional homodimer and two NOR molecules provides an idea of how these enzymes interact in cells, while the structure may not reflect the one in cells due to the membrane topology. Subsequent all-atom molecular dynamics (MD) simulations of the enzyme complex model in a membrane and structure-guided mutagenesis suggested that a few interenzyme salt bridges and coulombic interactions of NiR with the membrane could stabilize the complex of one NiR homodimer and one NOR molecule and contribute to rapid NO decomposition in cells. The MD trajectories of the NO diffusion in the NiR:NOR complex with the membrane showed that, as a plausible NO transfer mechanism, NO released from NiR rapidly migrates into the membrane, then binds to NOR. These results help us understand the mechanism of the cellular control of the action of cytotoxic NO.is a diffusible radical gas molecule that has been recognized as an integral signaling molecule in eukaryotes and bacteria (1, 2). NO plays pivotal roles in vasodilation, smooth muscle relaxation, neurotransmission, and the immune system in mammals. In bacteria, NO is also involved in several biological processes such as biofilm formation, quorum sensing, and symbiosis. NO is synthesized from L-arginine by an enzyme called nitric oxide synthase (NOS). In mammals, NO activates an NO receptor, soluble guanylate cyclase (sGC), upon the binding to heme in sGC, which induces the formation of signaling molecule, cGMP, from GTP. Currently, an NO binding protein homologous to the heme domain of sGC is thought to participate in bacterial NO signaling pathways (2). However, NO is a highly cytotoxic gas and easily reacts with biomolecules, despite its essential function. Thus, regulation of the cellular action of NO is crucial.Some bacteria would be exposed to large amounts of NO during denitrification, which implies that the control of NO dynamics is indispensable to minimize the cytotoxic effects of NO in such bacteria. Denitrification is a form of microbial anaerobic respiration by bacteria living in oxygen-limited environments in which the sequential reduction of nitrate to dinitrogen (NO 3 − → NO 2 − → NO → N 2 O → N 2 ) is coupled to bioenergy production. Denitrification is also crucial for the survival of some pathogenic bacteria inside host cells (3, 4). For example, Pseudomonas aeruginosa is a major opportunistic pathogen that survives through denitrification in hypoxic and anoxic environments such as the lungs of cystic fibros...

Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

No abstract