title: Soluble CuA domain of cyanobacterial cytochrome c oxidase.
abstract: The genomes of several cyanobacteria show the existence of gene clusters encoding subunits I, II, and III of aa(3)-type cytochrome c oxidase. The enzyme occurs on both plasma and thylakoid membranes of these oxygenic phototrophic prokaryotes. Here we report the expression and purification of a truncated subunit II copper A (Cu(A)) domain (i.e. the electron entry and donor binding site) of cytochrome c oxidase from the cyanobacterium Synechocystis PCC 6803 in high yield. The water-soluble purple redox-active bimetallic center displays a relatively low standard reduction potential of 216 mV. Its absorption spectrum at pH 7 is similar to that of other soluble fragments from aa(3)-type oxidases, but the insensitivity of both absorbance and circular dichroism spectra to pH suggests that it is less exposed to the aqueous milieu compared with other Cu(A) domains. Oxidation of horse heart cytochrome c by the bimetallic center follows monophasic kinetics. At pH 7 and low ionic strength the bimolecular rate constant is (2.1 +/- 0.3) x 10(4) m-1 s(-1), and the rates decrease upon the increase of ionic strength. Sequence alignment and modeling of cyanobacterial Cu(A) domains show several peculiarities such as: (i) a large insertion located between the second transmembrane region and the putative hydrophobic cytochrome c docking site, (ii) the lack of acidic residues shown to be important in the interaction between cytochrome c and Paracoccus Cu(A) domain, and (iii) an extended C terminus similar to Escherichia coli ubiquinol oxidase.
authors.affiliation: Institute of Physical Chemistry, Molecular Bioenergetics Group, University of Vienna, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Paumann
authors.forename: Borjana
authors.initials: B
authors.lastname: Lubura
authors.forename: Günther
authors.initials: G
authors.lastname: Regelsberger
authors.forename: Markus
authors.initials: M
authors.lastname: Feichtinger
authors.forename: Gunda
authors.initials: G
authors.lastname: Köllensberger
authors.forename: Christa
authors.initials: C
authors.lastname: Jakopitsch
authors.forename: Paul G
authors.initials: PG
authors.lastname: Furtmüller
authors.forename: Günter A
authors.initials: GA
authors.lastname: Peschek
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry; D000458:Cyanobacteria / Q000378:metabolism*; D045304:Cytochromes c / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D009206:Myocardium / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010230:Paracoccus / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D020524:Thylakoids / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D007477:Ions; D003300:Copper; D045304:Cytochromes c; D010088:Oxidoreductases; C432586:ubiquinol oxidase; D003576:Electron Transport Complex IV; D010100:Oxygen