title: Production, purification and detergent exchange of isotopically labeled Bacillussubtilis cytochrome b₅₅₈ (SdhC).
abstract: Cytochrome b₅₅₈ of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b₅₅₈. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b₅₅₈ produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature.
authors.affiliation: Microbiology Group, Department of Biology, Lund University, Sweden. michael.baureder@biol.lu.se
authors.forename: Michael
authors.initials: M
authors.lastname: Baureder
authors.forename: Lars
authors.initials: L
authors.lastname: Hederstedt
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009587:Nitrogen Isotopes / Q000737:chemistry; D010957:Plasmids / Q000235:genetics
chemical_list: D003573:Cytochrome b Group; D003902:Detergents; D009587:Nitrogen Isotopes; C036888:cytochrome b558; D019255:NADPH Oxidases