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Distal-to-Proximal NO Conversion in Hemoproteins: The Role of the Proximal Pocket

DOI: 10.1016/j.jmb.2010.10.035 DOI Help
PMID: 21073879 PMID Help

Authors: Mike Hough (University of Essex) , Svetlana Antonyuk (University of Liverpool) , Sonia Barbieri (University of Liverpool) , Neil Rustage (University of Liverpool) , Alison Mckay (Oregon University) , Amy Servid (Oregon University) , Robert Eady (University of Liverpool) , Colin Andrews (Oregon University) , Samar Hasnain (University of Liverpool)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Molecular Biology

State: Published (Approved)
Published: November 2010
Diamond Proposal Number(s): 1215

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.

Journal Keywords: Amino; Binding; Cytochromes; Ferrous; Kinetics; Models; Chemical; Mutagenesis; Site-Directed; Mutation; Nitric; Protein; Recombinant Proteins

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography