Kinetics of interprotein electron transfer between cytochrome c 6 and the soluble Cu A domain of cyanobacterial cytochrome c oxidase

Cytochrome c 6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b 6 f to photosystem I. The Cu A 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 c 6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c 6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccm A‐H gene cluster) and the recombinant soluble Cu A 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) × 10 5 M −1 s −1 and (3.9 ± 0.1) × 10 6 M −1 s −1 , respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 °C. This corresponds to an equilibrium constant K eq of 0.085 in the physiological direction (Δ r G ′0 =6.1 kJ/mol). The reduction of the Cu A fragment by cytochrome c 6 is almost independent on ionic strength, which is in contrast to the reaction of the Cu A domain with horse heart cytochrome c , which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c 6 as mobile electron carrier in both cyanobacterial electron transport pathways.