Natural Resistance to Inhibitors of the Ubiquinol Cytochrome c Oxidoreductase of Rubrivivax gelatinosus : Sequence and Functional Analysis of the Cytochrome bc ₁ Complex

Abstract

Biochemical analyses of Rubrivivax gelatinosus membranes have revealed that the cytochrome bc 1 complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin. This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q 0 and Q i sites. Because the resistance to cytochrome bc 1 inhibitors is primarily related to the cytochrome b primary structure, the petABC operon encoding the subunits of the cytochrome bc 1 complex of Rubrivivax gelatinosus was sequenced. In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome b polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome bc 1 inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the b H and b L hemes, respectively. These characteristics imply different functional properties of the cytochrome bc 1 complex of this bacterium. The consequences of these structural features for the resistance to inhibitors and for the properties of R. gelatinosus cytochrome bc 1 are discussed with reference to the structure and function of the cytochrome bc 1 complexes from other organisms.Ubihydroquinone cytochrome c oxidoreductase (cyt bc 1 complex) is an integral membrane complex involved in energy transduction in a wide range of organisms. Anoxygenic photosynthetic bacteria contain a cyt bc 1 complex, which serves for both light-driven electron transfer and dark respiration. For Rhodobacter sphaeroides and Rhodobacter capsulatus this enzyme catalyzes electron transfer from ubiquinol to soluble ferricytochrome c coupled to proton translocation across the membrane, thus generating an electrochemical gradient used for ATP synthesis. Oxygenic photosynthetic organisms contain a similar complex, i.e., cyt b 6f , which functions as a plastoquinol plastocyanin oxidoreductase (12). Structural and functional similarities have been reported for both types of cyt bc (3, 9, 41). The cyt bc complex was found to be made up of at least three subunits: cyt b (or b 6 ), containing the two b-type hemes of low (b L ) and high (b H ) redox potentials, cyt c 1 (or f) containing a c-type heme, and the Rieske protein (ISP) containing the [2Fe-2S] cluster. In addition to subunits containing the prosthetic groups and cofactors, the mitochondrial cyt bc 1 complex contains other subunits (4,29,30,46,47).Photosynthesis and aerobic respiration are affected by inhibitors. Some of these molecules are specific to the cyt bc complex. In fact, in many species the ubiquinol-cyt c oxidoreductase activity was found to be sensitive to quinone analogs such as myxothiazol, stigmatellin, antimycin, hydroxyquinoline Noxide (HQNO), and diuron. Extensive kinetic and genetic studies (7,13,16,17,19,28) as well as recent structural data available from X-ray crystallography of...

Investigation of the mechanism of proton translocation by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria: does the enzyme operate by a Q-cycle mechanism?

“…Corresponding values for the quinols were calculated by an analogous method: upon reaction with NADH the decrease in quinone concentration is equal to the increase in quinol concentration, so that comparison of the HPLC peak areas from sets of experiments from the same stock solution defined the ratio of the molar absorption coefficients (see Table 1). Comparison with previously reported values shows good agreement for DQH 2 [28,38] but significant discrepancy with the single reported value for Q 2 H 2 [39]. However, this published value is not consistent with reported values from other quinols.…”

Genome Analysis of Structure–Function Relationships in Respiratory Complex I, an Ancient Bioenergetic Enzyme

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