title: Ligand, cofactor, and residue vibrations in the catalytic site of endothelial nitric oxide synthase.
abstract: A study of bovine endothelial nitric oxide synthase by Fourier transform infrared (FTIR) spectroscopy in the 1000-2500 cm(-)(1) range is reported. Binding of CO to the reduced enzyme gives two heme(II)-CO nu(C)(-)(O) stretches (1927 and 1904 cm(-)(1)) which appear to be in rapid equilibrium. Photolysis of this heme(II)-CO compound is accompanied by perturbation of the local fine structure around the catalytic site giving vibrational changes of protein backbone, substrate, amino acid residues, and cofactors, to which heme, substrate arginine, and catalytic site residues contribute. Possible assignments of vibrations to heme, substrate arginine, and catalytic site residues are discussed. The discussion of assignments is informed by known structures, absorbance frequencies, and extinction coefficients of residues and cofactors, analysis of H(2)O-D(2)O exchange effects, analysis of substrate (14)N-(15)N (guanidinium)-arginine exchange effects, and comparison with the nNOS isoform (which differs in the replacement of asparagine 368 with an aspartate within the substrate binding site). The FTIR data can be modeled on the known structure of the catalytic site and indicate the extent of modulation of vibrational modes upon photolysis of the CO compound.
authors.affiliation: School of Biology, University of St. Andrews, UK. wji@st-andrews.ac.uk
authors.forename: W John
authors.initials: WJ
authors.lastname: Ingledew
authors.forename: Susan M E
authors.initials: SM
authors.lastname: Smith
authors.forename: Y T
authors.initials: YT
authors.lastname: Gao
authors.forename: R J
authors.initials: RJ
authors.lastname: Jones
authors.forename: John C
authors.initials: JC
authors.lastname: Salerno
authors.forename: Peter R
authors.initials: PR
authors.lastname: Rich
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry; D020134:Catalytic Domain; D002417:Cattle; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D009584:Nitrogen / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000378:metabolism; D009590:Nitrogen Radioisotopes / Q000378:metabolism; D010782:Photolysis; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D003067:Coenzymes; D008024:Ligands; D009419:Nerve Tissue Proteins; D009587:Nitrogen Isotopes; D009590:Nitrogen Radioisotopes; C498113:guanidinoarginine; D006418:Heme; D002248:Carbon Monoxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D009584:Nitrogen