Kinetics of interprotein electron transfer between cytochrome c₆ and the soluble Cu_A domain of cyanobacterial cytochrome c oxidase

Abstract

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 ccmA-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) Â 10 5 M À1 s À1 and (3.9 % 0.1) Â 10 6 M À1 s À1 , 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 (D r G 00 ¼ 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.

Cyanobacteria Metal Interactions: Requirements, Toxicity, and Ecological Implications

“…Two important proteins in the thylakoid (plastocyanin and a c-type cytochrome oxidase) are Cu-requiring proteins. Moreover, the necessity of delivering this element to the thylakoid membranes imposes an extra Cu pathway in cyanobacteria that is absent from other prokaryots (Cavet et al 2003) and the metalloprotein cytochrome c 6 is thought to participate in this trafficking, delivering Cu to the soluble domain of cytochrome oxidase (Paumann et al 2004). The molecule of chlorophyll needs Mg as the central atom of its porphyrin ring (Reid & Hunter 2004) while the enzyme RuBisCo (Ribulose 1,5-bisphosphate carboxylase/oxygenase) depends indirectly upon Zn in order to act as carboxylase (Smith & Ferry 2000).…”

Terminal oxidases of cyanobacteria

“…This has been reviewed by Peschek et al [8,11]. Recent work has used a soluble CtaC protein, which, within limits, has enabled estimation of the rate constants for the interaction between the donor and the Cu A centre, and will facilitate further probing of the role of specific residues through mutagenesis studies [18][19][20].…”

“…This is consistent with the notion that the hydrophobic N-terminus of Cyt c M may function as a transmembrane region rather than a targeting sequence. Although studies with soluble CtaC indicate that Cyt c M is not absolutely required for electron transfer [18][19][20], it may promote binding or act as a redox mediator between soluble electron carriers and COX [26]. Although the measured redox potential (E m ) of soluble Cyt c M was only 150 mV [27] (which would raise a problem for its reduction by Cyt f (cytochrome f ), E m ≈ 330 mV), membrane localization and/or the presence of other COX subunits could potentially increase this to allow Cyt c M to act as a redox mediator.…”

Cytochrome c 6 of Cyanobacteria and Algae: From the Structure to the Interaction

“…The electron flow in the Nostoc Cc 6 -CcO system is two to three orders of magnitude higher than that observed for the interaction of Synechocystis sp. PCC 6803 Cc 6 with the Cu A -binding domain of the CcO subunit II (Paumann et al 2004). Of particular interest is the fact that the electron transfer constant of the Nostoc Cc 6 -CcO system resembles the constant recently estimated in human mitochondrial complexes between Cc and CcO (Rodríguez-Roldán et al 2008).…”

“…The oxidation of Cc 6 by CcO in Synechocystis occurs without the formation of any electrostatically governed and kinetically detectable complex between these proteins (Paumann et al 2004). On the contrary, the Nostoc Cc 6 -CcO adduct is impaired by added salt, which reveals the existence of strong electrostatic attractive interactions, as previously reported for other respiratory Cc and CcO systems (Hazzard et al 1991; (Pettersen et al 2004)).…”