Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase.
Cytochrome c6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b6f to photosystem I. The CuA domain of cytochrome c oxidase, the terminal enzyme which catalyzes the four-electron reduction of molecular oxygen in the respiratory chains of mitochondria and many bacteria, also has a periplasmic location. In order to test whether cytochrome c6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccmA-H gene cluster) and the recombinant soluble CuA domain (i.e., the donor binding and electron entry site) of subunit II of cytochrome c oxidase from Synechocystis PCC 6803. The forward and the reverse electron transfer reactions were studied by the stopped-flow technique and yielded apparent bimolecular rate constants of (3.3 +/- 0.3) x 10(5) M(-1) s(-1) and (3.9 +/- 0.1) x 10(6) M(-1) s(-1), respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 degrees C. This corresponds to an equilibrium constant Keq of 0.085 in the physiological direction (DeltarG'0 = 6.1 kJ/mol). The reduction of the CuA fragment by cytochrome c6 is almost independent on ionic strength, which is in contrast to the reaction of the CuA domain with horse heart cytochrome c, which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c6 as mobile electron carrier in both cyanobacterial electron transport pathways.
Department of Physical Chemistry, Molecular Bioenergetics Group, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria.
Martina
M
Paumann
Markus
M
Feichtinger
Margit
M
Bernroitner
Judith
J
Goldfuhs
Christa
C
Jakopitsch
Paul G
PG
Furtmüller
Günther
G
Regelsberger
Günter A
GA
Peschek
Christian
C
Obinger
D000818:Animals; D003300:Copper / Q000737:chemistry; D000458:Cyanobacteria / Q000201:enzymology*; D045365:Cytochromes c6 / Q000737:chemistry* / Q000302:isolation & purification; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D009206:Myocardium / Q000201:enzymology; D009994:Osmolar Concentration; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D045365:Cytochromes c6; D011994:Recombinant Proteins; D003300:Copper; D003576:Electron Transport Complex IV