title: High reductase activity of recombinant NOS2 flavoprotein domain lacking the calmodulin binding regulatory sequence.
abstract: Nitric oxide synthases (NOSs) represent a special case of the cytochrome P450: cytochrome P450 reductase system in which the two redox partners occur as distinct domains on the same polypeptide chain and are linked by a calmodulin binding regulatory sequence. We expressed the carboxy-terminal, flavoprotein domain (residues 527-1144) of murine NOS2 in E. coli. The UV-visible spectrum of this domain resembles those of other flavoproteins, and the protein catalyses the reduction of ferricytochrome c by NADPH [Vmax = 3.1 +/- .1 mol cytochrome reduced/minute/mmol flavoprotein domain, Km (cytochrome c) = 23 +/- 2 microM, Km (NADPH) 0.30 +/- .06 microM]. The high reductase activity of this NOS2 flavoprotein domain, which lacks a calmodulin binding site, precludes a role for this site in mediating electron transfer within the flavoprotein domain of the intact enzyme. This is in contrast to the case of NOS1 and suggests that electron transfer is regulated differently in the two isoforms.
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: S
authors.initials: S
authors.lastname: Rafferty
authors.forename: H L
authors.initials: HL
authors.lastname: Malech
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D017931:DNA Primers; D005420:Flavoproteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D019001:Nitric Oxide Synthase; D009245:NADH Dehydrogenase