Neuronal nitric oxide synthase ligand and protein vibrations at the substrate binding site. A study by FTIR.
Improvements in sensitivity and data processing of Fourier transform infrared (FTIR) spectroscopy enable it to be used to detect changes in protein structure at the atomic level. This paper reports a study of neuronal nitric oxide synthase (nNOS) by FTIR difference spectroscopy in the 1000-2500 cm(-1) range where vibrational bands of ligands, prosthetic groups, and protein and amino acid side chains are found. We have exploited the photolyzable CO compound of the ferrous heme of nNOS to produce light-induced CO photolysis difference spectra and to compare spectra after hydrogen/deuterium exchange. In (reduced) minus (reduced plus CO) difference spectra, negative bands at 1931 and 1907 cm(-1) are observed due to photolysis of multiple forms of ferrous heme-ligated CO, similar to those observed by resonance Raman spectroscopy [Wang et al. (1997) Biochemistry 36, 4595-4606]. Photolysis of the ferrous heme CO compound is accompanied by hitherto unreported changes in the 1000-2000 cm(-1) region that arise from changes of protein backbone, substrate, amino acid side chain, and cofactor vibrations. Preliminary assignments of vibrations are made on the basis of frequencies and the effects of hydrogen/deuterium exchange, and in the light of known atomic structures.
Ingledew
W John
WJ
School of Biology, University of St. Andrews, St. Andrews KY16 9JF, UK. wji@st-andrews.ac.uk
Smith
Susan M E
SM
Salerno
John C
JC
Rich
Peter R
PR
D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010782:Photolysis; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014732:Vibration
D011994:Recombinant Proteins; D002248:Carbon Monoxide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat