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Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 H + e − with uptake of 1 H + e − from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K+ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c2-oxidoreductase showed a pumping activity of 0.9 H + e − , which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.

Three plasmids containing the transposon Tn5, i.e. pSUP201::Tn5, pACYC184::Tn5 and pJB4JI were transferred from Escherichia coli to Rhodopseudomonas capsulata in order to mutagenize the genome. Mutants defective in bacteriochlorophyll and carotenoid synthesis and mutants unable to form the photochemical reaction center or one of the light-harvesting complexes were isolated. Of special interest were mutants that could not form the light-harvesting complex B800-850. Two of these mutants synthesized only two of the three polypeptides of this complex whereas the corresponding near infrared absorbance bands were not observed. Complementation analysis with the Rprime plasmid pRPS404, which contains a 50 kb region of the genome of R. capsulata carrying most genes responsible for expression of photosynthetic apparatus, revealed that some genes of the B800-850 light-harvesting complex lie outside this photosynthetic gene cluster.

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c2 from mutant H41 demonstrated at the same time the participation of the c2-polypeptide in the regulation of cytochrome c oxidase.

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c′ into the medium and lack cytochrome c 2.

The role of cytochromes in the electron-transport pathway to trimethylamine N -oxide (TMAO)/dimethylsulphoxide (DMSO) reductase in the photosynthetic bacterium Rhodobacter capsulatus was investigated. Reduced-minus-oxidized difference spectra in intact cells with TMAO or DMSO as oxidant revealed cytochrome absorbance changes with a maximum at 559 nm and a shoulder between 548 nm and 556 nm. The former change indicates a role for a 6-type cytochrome and the latter for a c-type cytochrome, both of which are distinct from the cytochrome bc 1 complex. Cytochrome c -556 was identified in a bacterial periplasmic fraction as a redox component which couldbe oxidised by TMAO or DMSO. Cytochrome c -556 was the only cytochrome species which co-fractionated with TMAO/DMSO reductase following gel filtration of a post-chromatophore supernatant produced after French presstreatment of intact cells. The mid-point redox potential (pH 7.6) of cytochrome c -556 was + 105 mV ( n = 1). It is suggested that TMAO/DMSO reductase and cytochrome c -556 form a structural and functional association in the periplasm of Rhodobacter capsulatus .

A cytochrome oxidase (EC 1.9.3.1) was solubilized from the membrane fraction of aerobically grown cells of Rhodopseudomonas capsulata by treatment with Triton X-100. The enzyme was purified 160 fold by chromatography on DEAE-Sepharose CL-6B and affinity chromatography on cytochrome c-thiol activated Sepharose 4B. The purified enzyme has a pH-optimum at 8.5 and a temperature optimum at 35 °C. The ap­ parent Km for reduced horse cytochrome c is 24 μм (at pH 8 and 30 °C). The purified cytochrome oxidase was 50% inhibited by 1.5 μм KCN and 10 μм NaN3. The purified enzyme contained one polypeptide of mr 65,000 and 6-type cytochrome.

A cytochrome c (cyt. c ) was solubilized with Triton-X-100 and co-purified with cytochrome c oxidase from membranes of chemotrophically grown cells of Rhodopseudomonas capsulata . Cyt. c and cytochrome oxidase were separated on Sephadex G-50 columns. Antibodies against cytochrome c 2 from the same bacterium did not cross react with the membrane-bound cyt. c . The IEP of the membrane-bound cyt. c was found to be pH 8.2 the midpoint potential was 234 ± 11 mV at pH 7.0. This cyt. c binds CO. The native cyt. c is a dimer with an apparent M r of 25 000 containing 2 mol heme per mol dimer, which is believed to function as an electron donor for the high-potential cytochrome c oxidase.