title: One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli and demonstration that associated quinone is not required for the structural integrity of the oxidase.
abstract: The cytochrome bo3 ubiquinol oxidase from Escherichia coli is a member of the heme-copper superfamily of proton-pumping respiratory oxidases. An improved preparative protocol was desired that would minimize the potential damage during protein isolation of labile mutants of the oxidase. Variants of the oxidase containing a histidine tag at the carboxy-terminus of either subunit I, II or III were constructed. The constructs with the histidine tag on either subunit I or II successfully allowed the enzyme to be isolated with high purity in one step using Ni2+ affinity chromatography. The enzyme with the histidine tag on subunit II is particularly useful insofar as the enzyme isolated in this manner has little, if any, heterogeneity resulting from the presence of heme O in the low spin heme-binding site, i.e., cytochrome oo3 is minimized. The enzyme can be prepared in virtually any quantity very rapidly and is suitable for biophysical characterization. Cytochrome bo3 was prepared in either Triton X-100, sucrose monolaurate, or dodecyl maltoside. The enzyme isolated in the presence of either sucrose monolaurate or dodecyl maltoside contains approximately one equivalent of associated ubiquinone, whereas this is absent when Triton X-100 is used. However, the UV/vis absorbance and steady-state kinetic properties of the enzyme are virtually identical regardless of which detergent is used. These data are consistent with previous reports that cytochrome bo3 contains an equivalent of 'tightly associated' ubiquinone, but clearly demonstrate that this quinone can be removed without damaging the enzyme and is not critical to the maintenance of the native structure of the oxidase.
authors.affiliation: School of Chemical Sciences, University of Illinois, Urbana 61801, USA.
authors.forename: J N
authors.initials: JN
authors.lastname: Rumbley
authors.forename: E
authors.initials: E
authors.lastname: Furlong Nickels
authors.forename: R B
authors.initials: RB
authors.lastname: Gennis
mesh_terms: D016227:Benzoquinones / Q000032:analysis / Q000737:chemistry*; D001665:Binding Sites; D002846:Chromatography, Affinity; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005960:Glucosides; D006418:Heme / Q000032:analysis / Q000378:metabolism; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009532:Nickel; D017830:Octoxynol; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D013395:Sucrose / Q000031:analogs & derivatives
chemical_list: D016227:Benzoquinones; D003573:Cytochrome b Group; D003580:Cytochromes; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; C083850:cytochrome bo3, E coli; C018152:sucrose monolaurate; C004532:quinone; D006418:Heme; D006639:Histidine; D013395:Sucrose; C040358:dodecyl maltoside; D009532:Nickel; D017830:Octoxynol; D004262:DNA Restriction Enzymes