title: Structures of reduced and ligand-bound nitric oxide reductase provide insights into functional differences in respiratory enzymes.
abstract: Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N2O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non-heme Fe center. We report herein on the structures of the reduced and ligand-bound forms of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3-2.7 Å, to elucidate structure-function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO-bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH3-CH=N-OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ∼0.5 Å increase in the heme/non-heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton-pumping activity in cNOR, because redox-coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non-heme Fe distance even in the bulky ligand-bound form of cNOR (∼4.5 Å) than the heme/Cu distance in CCO (∼5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N-N coupling to produce a hyponitrite intermediate for the generation of N2O.
authors: Biometal Science Laboratory, RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan; Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors: Nozomi
authors: N
authors: Sato
authors: Shoko
authors: S
authors: Ishii
authors: Hiroshi
authors: H
authors: Sugimoto
authors: Tomoya
authors: T
authors: Hino
authors: Yoshihiro
authors: Y
authors: Fukumori
authors: Yoshihiko
authors: Y
authors: Sako
authors: Yoshitsugu
authors: Y
authors: Shiro
authors: Takehiko
authors: T
authors: Tosha
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D008024:Ligands; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D010091:Oximes / Q000737:chemistry / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D010091:Oximes; D002248:Carbon Monoxide; D010088:Oxidoreductases; C034747:nitric-oxide reductase; C023728:acetaldehyde oxime
keywords: X-ray crystallography; cytochrome c oxidase; heme; iron-containing enzyme; proton transfer; resonance Raman