title: Integration of the thylakoid membrane protein cytochrome b6 in the cytoplasmic membrane of Escherichia coli.
abstract: An overexpression system for spinach apocytochrome b(6) as a fusion protein to a maltose-binding protein in Escherichia coli was established using the expression vector pMalp2. The fusion of the cytochrome b(6) to the periplasmic maltose-binding protein directs the cytochrome on the Sec-dependent pathway. The cytochrome b(6) has a native structure in the bacterial cytoplasmic membrane with both NH(2) and COOH termini on the same, periplasmic side of the membrane but has the opposite orientation compared to that in thylakoid. Our data also show that in the E. coli cytoplasmic membrane, apocytochrome b(6) and exogenic hemes added into a culture media spontaneously form a complex with similar spectroscopic properties to native cytochrome b(6). Reconstituted membrane-bound cytochrome b(6) contain two b hemes (alpha band, 563 nm; average E(m,7) = -61 +/- 0.84 and -171 +/- 1.27 mV).
authors.affiliation: The Institute of Biochemistry and Molecular Biology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland.
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Króliczewski
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Hombek-Urban
authors.forename: Andrzej
authors.initials: A
authors.lastname: Szczepaniak
mesh_terms: D001051:Apoenzymes / Q000096:biosynthesis / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D003080:Cold Temperature; D045347:Cytochromes b6 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology* / Q000378:metabolism; D004563:Electrochemistry; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000032:analysis / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology* / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010957:Plasmids; D013053:Spectrophotometry; D018724:Spinacia oleracea; D013194:Staining and Labeling; D020524:Thylakoids / Q000201:enzymology* / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D002352:Carrier Proteins; D045347:Cytochromes b6; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D006418:Heme