Isolation and Characterization of a Two-Subunit Cytochrome b−c₁ Subcomplex from Rhodobacter capsulatus and Reconstitution of Its Ubihydroquinone Oxidation (Q_o) Site with Purified Fe-S Protein Subunit

Abstract

The presence of a two-subunit cytochrome (cyt) b-c1 subcomplex in chromatophore membranes of Rhodobacter capsulatus mutants lacking the Rieske iron-sulfur (Fe-S) protein has been described previously [Davidson, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351-3358]. Here, this subcomplex was purified to homogeneity in large quantities, and its properties were characterized. As expected, it contained stoichiometric amounts of cyt b and cyt c1 subunits forming a stable entity devoid of the Fe-S protein subunit. The spectral and thermodynamic properties of its heme groups were largely similar to those of a wild-type bc1 complex, except that those of its cyt bL heme were modified as revealed by EPR spectroscopy. Dark potentiometric titrations indicated that the redox midpoint potential (Em7) values of cytochromes bH, bL, and c1 were very similar to those of a wild-type bc1 complex. The purified b-c1 subcomplex had a nonfunctional ubihydroquinone (UQH2) oxidation (Qo) site, but it contained an intact ubiquinone (UQ) reductase (Qi) site as judged by its ability to bind the Qi inhibitor antimycin A, and by the presence of antimycin A sensitive Qi semiquinone. Interestingly, its Qo site could be readily reconstituted by addition of purified Fe-S protein subunit. Reactivated complex exhibited myxothiazol, stigmatellin, and antimycin A sensitive cyt c reductase activity and an EPR gx signal comparable to that observed with a bc1 complex when the Qo site is partially occupied with UQ/UQH2. However, a mutant derivative of the Fe-S protein subunit lacking its first 43 amino acid residues was unable to reactivate the purified b-c1 subcomplex although it could bind to its Qo site in the presence of stigmatellin. These findings demonstrated for the first time that the amino-terminal membrane-anchoring domain of the Fe-S protein subunit is necessary for UQH2 oxidation even though its carboxyl-terminal domain is sufficient to provide wild-type-like interactions with stigmatellin at the Qo site of the bc1 complex.

A semiquinone intermediate generated at the Q o site of the cytochrome bc 1 complex: Importance for the Q-cycle and superoxide production

“…The resulting cyt bc 1 complex is identical to the wild type except that the Rieske subunit is not incorporated (38,39). Fig.…”

“…As an additional test of whether this signal truly originates from the Q o site of the cyt bc 1 complex, we performed experiments under conditions identical to those described above using purified cyt bc 1 from a strain of R. capsulatus in which the primary oxidant to UQH 2 within the cyt bc 1 complex, the Rieske 2Fe2S cluster, is eliminated through mutation of a histidine (H135) ligand to the 2Fe2S cluster to leucine (H135L) (38). The resulting cyt bc 1 complex is identical to the wild type except that the Rieske subunit is not incorporated (38,39).…”

Movement of the Iron−Sulfur Head Domain of Cytochrome bc1 Transiently Opens the Catalytic Qo Site for Reaction with Oxygen

“…Wild-type and mutated cytochrome bc 1 were isolated from the appropriate strains (25)(26)(27) of Rhodobacter capsulatus as described previously (28). Mutated cytochrome bc 1 included the following cofactor knockout forms: the c 1 knockout with the M183L mutation in cytochrome c 1 (26) and the FeS motion knockout with the +2Ala insertion in the neck region of the FeS subunit (27 enzymatic activity of cytochrome bc 1 was assayed by measuring the DBH 2 (2,3-dimethoxy-5-decyl-6-methyl-1,4-benzohydroquinone)-dependent reduction of mitochondrial horse cytochrome c as described previously (25).…”

Cytochrome bc1-cy Fusion Complexes Reveal the Distance Constraints for Functional Electron Transfer Between Photosynthesis Components

“…The R. capsulatus cyt bc 1 complex was purified as described previously (22). Protein concentrations were determined using the bicinchoninic acid method (23) with bovine serum albumin as a standard.…”

“…Earlier, we partially purified the cyt bc 1 -c y fusion complex (19) using the procedure described by Valkova-Valchanova et al (22). However, as detailed characterization required purer samples, we developed a new procedure.…”