A Cytochrome b/c₁ Complex with Ubiquinol–Cytochrome c₂ Oxidoreductase Activity from Rhodopseudomonas sphaeroides GA

Abstract

A cytochrome b/cl complex which catalyses the reduction of cytochrome c by ubiquinol has been isolated from Rhodopseudomonas sphaeroides GA. It contains two hemes b and substoichiometric amounts of ubiquinone-30 and of the Rieske Fe-S center per cytochrome c1, and is essentially free of reaction center and bacteriochlorophyll. The complex consists of three major polypeptides with apparent molecular masses of 40, 34 and 25 kDa. The 34-kDa polypeptide carries heme. Cytochrome c1 has a midpoint potential of 285 mV. For cytochrome b two midpoint potentials, at 50 and -60 mV, at pH 7.4, can be derived if one assumes two components of equal amount. Ubiquinol -cytochrome c oxidoreductase activity is specific for ubiquinol and bacterial cytochromes c, and is inhibited by antimycin A and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. The complex shows oxidant-induced reduction of cytochrome b.Respiratory as well as photosynthetic electron transport, in mitochondria [l] (Krinner et al., unpublished). Compared to the mitochondria1 complexes the latter two represent relatively simple polypeptide structures. Preparations from anoxygenic photosynthetic bacteria have not been reported. They would be desirable not only for comparative aspects, but also as a biochemical complimentation to the detailed flash spectrophotometric studies, especially on membranes from Rhodospirillaceae [3 -51.By adotping the procedure for the purification of the cytochrome flb6 complex from spinach chloroplasts [2] we succeeded in the isolation of an active cytochrome b/cl complex from Rhodopseudomonas sphaeroides GA, which is presented here. The procedure is based on the use of octylglucoside/ cholate mixtures to solubilize the complex, which has been also successfully employed to isolate coupling factor ATPases [12,13]. MATERIALS AND METHODS Preparation of Ubiquinol-Cytochrome c2 OxidoreductaseCells of Rhodopseudomonas sphaeroides GA were grown and harvested, and chromatophores were prepared thereDedicated to the memory of Assunta Baccarini-Melandri who initiated this work. Other MaterialsUbiquinols were kindly donated by Hoffmann-LaRoche, Basel. Cytochrome c2 from R. sphaeroides was prepared according to Bartsch [15], and cytochrome c from Saccharomyces cerevisiae was obtained from Sigma. All other sources for materials are given in [2]. AssaysActivity of ubiquinol -cytochrome c2 oxidoreductase was measured in an Aminco DW-2 spectrophotometer as described

Evidence for cytochrome bc1 complex involvement in nitrite reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

“…nmol cytochrome CT'. h-l, which is about 60% of that reported in [2]. These results indicated that the isolated cytochrome bc, complex is comparable to that of R. sphaeroides with respect to cytochrome composition and cytochrome c2 reduction activity.…”

Two distinct heterodisulfide reductase‐like enzymes in the sulfate‐reducing archaeon Archaeoglobus profundus

“…The absorbance maxima at 426 nm (c band), 530 nm (b band) and a split a band at 557 nm and 562 nm are characteristic for hemes of the b-type [26]. A similar splitting of the a band has been observed for other heme proteins, for example the cytochrome b L of the cytochrome bc 1 complex form Rhodopseudomonas sphaeroides GA [27]. Pyridine hemochrome reduced-oxidized difference spectra show maxima for the a and b band at 553 and 521 nm.…”

Structure and Function of CytochromebcComplexes

“…A procedure based on salt fractionation and sucrose density gradient centrifugation of octyl glucoside extracts was introduced by Hauska and coworkers for purification of the chloroplast b 6 f complex (45), and was adapted to isolation of the bc 1 complex from Rhodobacter (46) and the b 6 f complex of cyanobacteria (47). Clark & Hind (48) purified spinach b 6 f using a column of immobilized cytochrome c (horse).…”