Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS).
Nitric oxide synthase (NOS) catalyzes the NADPH- and O(2)-dependent oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline via an N(G)-hydroxy-l-arginine (NHA) intermediate. Mammalian NOSs have been studied quite extensively; other eukaryotes and some prokaryotes appear to express NOS-like proteins comparable to the oxygenase domain of mammalian NOSs. In this study, a recombinant NOS-like protein from the thermostable bacterium Geobacillus stearothermophilus (gsNOS) has been characterized using magnetic circular dichroism (MCD) and UV-Vis absorption spectroscopic techniques. Spectral comparisons of ligand complexes (with O(2), NO and CO) of substrate-bound (l-Arg or NHA) gsNOS, including the key oxyferrous complex studied at -50 degrees C in cryogenic mixed solvents, with analogous mammalian NOS complexes indicate overall spectroscopic similarities between gsNOS and mammalian NOSs. However, more detailed spectral comparisons reflect subtle structural differences between gsNOS and mammalian NOSs. This may be due to an incomplete tetrahydrobiopterin (BH(4))-binding site and low BH(4)-binding affinity, which may become even lower in the presence of cryosolvent in gsNOS. Although BH(4)-binding may be altered, gsNOS appears to require the pterin for NO production since formation of the stable ferric-NO product complex was only observed when excess BH(4) (>150muM) over gsNOS was present upon single turnover reaction in which O(2) was bubbled into dithionite-reduced NHA-bound protein solution at -35 degrees C or -50 degrees C.
Kinloch
Ryan D
RD
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, United States. rdkinloch@gmail.com
Sono
Masanori
M
Sudhamsu
Jawahar
J
Crane
Brian R
BR
Dawson
John H
JH
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism / Q000379:methods*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D009569:Nitric Oxide; D007501:Iron; D019001:Nitric Oxide Synthase; D010100:Oxygen