Rhodobacter sphaeroides contains at least two different cytochrome c oxidases. When these bacteria are grown with high aeration, the traditional aa3-type cytochrome c oxidase is present at relatively high levels. However, under microaerophilic growth conditions or when the bacteria are grown photosynthetically, the amount of the aa3-type oxidase is greatly diminished and an alternate cytochrome c oxidase is evident. This alternate oxidase has been purified and characterized. The enzyme consists of three subunits by SDS-PAGE analysis (Mapp 45, 35, and 29 kDa). Two of the three subunits (Mapp 35 and 29 kDa) contain covalently bound heme C. Metal and heme analyses indicate that the oxidase contains heme C, heme B (protoheme IX), and Cu in a ratio of 3:2:1. Cryogenic Fourier transform infrared (FTIR) difference spectroscopy of the CO adduct of the reduced enzyme shows that the oxidase contains a heme-copper binuclear center and, thus, is a member of the heme-copper oxidase superfamily. In contrast to other members of this superfamily, however, this oxidase does not contain either heme O or heme A as a component of the binuclear center, but has heme B at this site. The single equivalent of Cu found in the oxidase is accounted for by the CuB component at the binuclear center. This suggests that this oxidase does not contain CuA, which is found in all other well-characterized cytochrome c oxidases. Both EPR and optical spectroscopic studies are consistent with this conclusion, also indicating that this oxidase does not contain CuA.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.

Amino acid sequence data have revealed that the bo-type ubiquinol oxidase from Escherichia coli is closely related to the eukaryotic aa3-type cytochrome c oxidases. In the cytochrome c oxidases, the reduction of oxygen to water occurs at a binuclear center comprised of heme a3 and Cu(B). In this paper, Fourier transform infrared (FTIR) spectroscopy of CO bound to the enzyme is used to directly demonstrate that the E. coli bo-type ubiquinol oxidase also contains a heme-copper binuclear center. Photolysis of CO ligated to heme o at low temperatures (e.g., 30 K) results in formation of a CO-Cu complex, showing that there is a heme-Cu(B) binuclear center similar to that formed by heme a3 and Cu(B) in the eukaryotic oxidase. It is further demonstrated that the cyoE gene product is required for the correct assembly of this binuclear center, although this polypeptide is not required as a component of the active enzyme in vitro. The cyoE gene product is homologous to COX10, a nuclear gene product from Saccharomyces cerevisiae, which is required for the assembly of yeast cytochrome c oxidase. Deletion of the cyoE gene results in an inactive quinol oxidase that is, however, assembled in the membrane. FTIR analysis of bound CO shows that Cu(B) is present in this mutant but that the heme-Cu(B) binuclear center is abnormal. Analysis of the heme content of the membrane suggests that the cyoE deletion results in the insertion of heme B (protoheme IX) in the binuclear center, rather than heme O.(ABSTRACT TRUNCATED AT 250 WORDS)

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The quinol oxidase, cytochrome bd, functions as a terminal oxidase in the Escherichia coli respiratory chain, reducing O 2 to water and using ubiquinol-8 or menaquinol-8 as its immediate reductant. The oxidation of quinol is by the low-spin ferri-haem, cytochrome b 558. This occurs at a quinol-binding site by sequential one electron steps, requiring the stabilisation of the semiquinone intermediate. We have observed, by EPR spectroscopy, the properties of this semiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess of ubiquinone-8 and menaquinone-8 analogues. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a semiquinone anion of this type. The line shape is Gaussian. The semiquinone is highly stabilised with respect to free semiquinone; significant free radical can be observed at pH 7.0 and above. The pH dependence of the redox reactions indicate that the anionic form of the semiquinone and the neutral form of the quinol predominate in the pH range studied. The pH dependence of the mid-point potentials of the one electron reactions from pH 7.0Ϫ9.0 is 120 mV/pH change for the semiquinone anion to quinol (E 2) and none for the quinone to semiquinone (E 1 ). The semiquinone radical is attenuated on titration with putative inhibitors of this quinone-binding site. We have similarly studied the semiquinone in membrane preparations from a strain with overexpression of cytochrome bd oxidase. The data can be fitted with the assumption of a single quinone-binding site.Keywords : cytochrome bd; quinol oxidation; quinone-binding site; semiquinone stabilisation.Quinone-binding sites are obligatory in quinone-utilising re-mitochondria and chloroplasts. The respiratory chains of Escherichia coli contain them because the respiratory chain is termispiratory and photosynthetic complexes. In addition to their importance in catalysis in electron-transfer systems involved in en-nated by quinol oxidases ; cytochromes bo and bd, fumarate reductase, dimethylsulphoxide reductase and nitrate reductase. In ergy transduction, these loci are the site of action of numerous inhibitors including pesticides, herbicides and antibiotics [1].the Escherichia coli respiratory chain two quinones are employed, ubiquinone-8 and menaquinone-8. Most dehydrogenases Quinone-binding sites are of three types; reduction (acceptor), oxidation (donor) and pair-splitting loci. The site under con-and oxidases do not appear to discriminate markedly between them, although the relative concentrations of the two species are sideration here is an oxidation site as it supplies reducing equivalents to the low-spin b 558 of cytochrome bd [2] (but see also regulated and influenced by growth conditions [6].Mitochondria have acceptor loci associated with the NADH [3]); the potentials of the two half reactions thus have to be approximately equated as the electrons are fed to the same and succinate dehydrogenases. They also have two sites associated with complex III and involved in the proton-motive...