title: Distal-to-proximal NO conversion in hemoproteins: the role of the proximal pocket.
abstract: Hemoproteins play central roles in the formation and utilization of nitric oxide (NO) in cellular signaling, as well as in protection against nitrosative stress. Key to heme-nitrosyl function and reactivity is the Fe coordination number (5 or 6). For (five-coordinate) 5c-NO complexes, the potential for NO to bind on either heme face exists, as in the microbial cytochrome c' from Alcaligenes xylosoxidans (AxCYTcp), which forms a stable proximal 5c-NO complex via a distal six-coordinate NO intermediate and a putative dinitrosyl species. Strong parallels between the NO-binding kinetics of AxCYTcp, the eukaryotic NO sensor soluble guanylate cyclase, and the ferrocytochrome c/cardiolipin complex have led to the suggestion that a distal-to-proximal NO switch could contribute to the selective ligand responses in gas-sensing hemoproteins. The proximal NO-binding site in AxCYTcp is close to a conserved basic (Arg124) residue that is postulated to modulate NO reactivity. We have replaced Arg124 by five different amino acids and have determined high-resolution (1.07-1.40 Å) crystallographic structures with and without NO. These, together with kinetic and resonance Raman data, provide new insights into the mechanism of distal-to-proximal heme-NO conversion, including the determinants of Fe-His bond scission. The Arg124Ala variant allowed us to determine the structure of an analog of the previously unobserved key 5c-NO distal intermediate species. The very high resolution structures combined with the extensive spectroscopic and kinetic data have allowed us to provide a fresh insight into heme reactivity towards NO, a reaction that is of wide importance in biology.
authors.affiliation: Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, UK.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hough
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Antonyuk
authors.forename: Sonia
authors.initials: S
authors.lastname: Barbieri
authors.forename: Neil
authors.initials: N
authors.lastname: Rustage
authors.forename: Alison L
authors.initials: AL
authors.lastname: McKay
authors.forename: Amy E
authors.initials: AE
authors.lastname: Servid
authors.forename: Robert R
authors.initials: RR
authors.lastname: Eady
authors.forename: Colin R
authors.initials: CR
authors.lastname: Andrew
authors.forename: S Samar
authors.initials: SS
authors.lastname: Hasnain
mesh_terms: D000421:Alcaligenes / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009569:Nitric Oxide / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005296:Ferrous Compounds; D011994:Recombinant Proteins; D009569:Nitric Oxide; D045304:Cytochromes c