title: Expression and characterisation of a major c-type cytochrome encoded by gene kustc0563 from Kuenenia stuttgartiensis as a recombinant protein in Escherichia coli.
abstract: The purification of small quantities of a major small c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis has recently been reported. In order to characterise this protein further we have expressed the gene encoding this cytochrome in Escherichia coli and have purified the protein to homogeneity. The protein is directed to the E. coli periplasm using its native signal sequence suggesting that it may be translocated via a Sec-type system in K. stuttgartiensis. The cytochrome has the visible spectroscopic properties typical of a low-spin c-type cytochrome, but these spectroscopic features broaden in high salt solutions. The oxidised cytochrome was able to bind the ligands NO and cyanide. A redox potential of +230 mV suggests that the protein is suitable to act as an electron carrier protein that may be involved in the respiratory chain between hydrazine oxidation and the reduction of nitrite. The predicted protein sequence for the cytochrome suggests it to be a predominantly alpha-helical protein, and this is supported by circular dichroism.
authors.affiliation: Department of Biology (Area 10), University of York, Heslington, York YO10 5YW, UK. w.huston@qut.edu.au
authors.forename: Wilhelmina M
authors.initials: WM
authors.lastname: Huston
authors.forename: Harry R
authors.initials: HR
authors.lastname: Harhangi
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Leech
authors.forename: Clive S
authors.initials: CS
authors.lastname: Butler
authors.forename: Mike S M
authors.initials: MS
authors.lastname: Jetten
authors.forename: Huub J M
authors.initials: HJ
authors.lastname: Op den Camp
authors.forename: James W B
authors.initials: JW
authors.lastname: Moir
mesh_terms: D001421:Bacteria, Anaerobic / Q000201:enzymology*; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D010084:Oxidation-Reduction; D019897:Periplasm / Q000378:metabolism; D011199:Potentiometry; D013056:Spectrophotometry, Ultraviolet
chemical_list: D045304:Cytochromes c