John van der Oost

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa3‐type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa3‐type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem‐containing oxidase, called cytochrome bb3, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::KmR) an alternative cyto‐chrome c oxidase has been characterized; this cbb3‐type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox‐driven proton pumps. The proton‐pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.

Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators.

The nir and nor genes, which encode nitrite and nitric oxide reductase, lie close together on the DNA of Paracoccus denitrificans. We here identify an adjacent gene, nnr, which is involved in the expression of nir and nor under anaerobic conditions. The corresponding protein of 224 amino acids is homologous with the family of FNR proteins, although it lacks the N‐terminal cysteines. A mutation in the nnr gene had a negative effect on the expression of nitrite and nitric oxide reductase. Synthesis of membrane bound nitrate reductase, of nitrous oxide reductase, and of the cbb 3‐type cytochrome c oxidase were not affected by mutation of this gene. These results suggest that denitrification in P. denitrificans may be governed by a signal transduction network that is similar to that involved in oxygen regulation of nitrogen metabolism in other organisms.

Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design

In Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e coupling efficiencies of the cbb3‐type

The CuAsite of the caa3-type oxidase of Bacillus subtilis is a mixed-valence binuclear copper centre

A copper-containing domain of the caa3-type oxidase from Bacillus subtilis has been expressed as a water-soluble protein in the cytoplasm of Escherichia coli. Electron paramagnetic resonance (EPR) spectra of this purple domain show well-resolved lines in the gz resonance, both at X-band and S-band frequencies. Interpretation of EPR spectra and analytical data indicate a binuclear copper site consisting of one Cu2+ and one Cu1+. This copper site closely resembles CuA in subunit II of cytochrome c oxidase and is shown here to be a mixed-valence [Cu2+-Cu1+) binuclear centre.

Introduction of a CuA site into the blue copper protein amicyanin from Thiobacillus versutus

The C‐terminal loop of the blue copper protein amicyanin, which contains three of the four active site ligands, has been replaced with a CuA binding loop. The purple protein produced has visible and EPR spectra identical to those of a CuA centre. Recent evidence strongly suggests that the CuA centre of cytochrome c oxidase and the A centre of nitrous oxide reductase are similar and are both binuclear. It therefore follows that the purple amicyanin mutant created here also possesses a binuclear CuA centre.

Hydrogenase activity in nitrate-grown cells of the unicellular cyanobacterium Cyanothece PCC 7822

The activity of hydrogenase in intact cells of the unicellular cyanobacterium Cyanothece PCC 7822 was investigated using a mass spectrometer with a permeable membrane inlet. A small hydrogenase-catalyzed hydrogen production was observed with nitrate-grown cells under anoxic conditions in the dark. The same cells were also capable of a much greater rate of hydrogen uptake, induced by oxygen as well as light. Light-induced hydrogen uptake was inhibited by uncoupler. In contrast, addition of uncoupler caused a four-fold stimulation of anoxic hydrogen production in the dark. It is suggested that anoxic hydrogen production is the result of fermentative metabolism.Cyanobacteria are generally considered to have at least two distinct hydrogenases (Houchins 1984). One is a membrane-bound uptake hydrogenase which appears to be associated with nitrogen fixation, removing the hydrogen produced by nitrogenase with the concomitant production of reductant or ATP (Eisbrenner et al. 1978). The second is a reversible hydrogenase located in the cytoplasm and not closely linked to nitrogen metabolism. The reversible character of this enzyme can be demonstrated in the presence of suitable electron donors or acceptors; hydrogen consumption and evolution occur at similar rates (Lambert and Smith 1980).A reversible hydrogenase capable of reducing protons with the artificial electron donor couple dithionite and methyl viologen is widely distributed amongst cyanobacteria. However its physiological role remains unclear. The enzyme appears to be sensitive to oxygen, and consequently in vivo activity can only be demonstrated under anoxic conditions (Houchins 1984).On the basis of in vivo measurements with tritium and the observed low Km for hydrogen, the function of the reversible hydrogenase of the heterocystous cyanobacterium Anabaena has been proposed to be the uptake of hydrogen as a means of collecting additional reducing power during growth in light-limited anoxic environments (Spiller et al. 1983; Houchins 1984). However, Hallenbeck et al. (1981) reported a modest production of hydrogen by intact filaments of Anabaena.An example of a function of the reversible hydrogenase in the production of hydrogen is provided by the nonheterocystous filamentous cyanobacterium Oscillatoria limnetica. This organism is capable of shifting between oxygenic and anoxygenic photosynthesis (Oren and Padan 1978). In the latter case sulfide is the electron donor supporting photoreduction of CO2 via photosystem I only. However when CO2 is limiting, excess reducing equivalents are removed by a reversible hydrogenase (Belkin and Padan 1978). This hydrogen production probably enables the organism to continue photophosphorylation under these conditions.We recently reported that the unicellular cyanobacterium Cyanothece 7822 is capable of hydrogenase-catalyzed hydrogen production in vivo, without the addition of artificial reductants (Van der Oost et al. 1987). In this paper we have investigated the in vivo activity of the hydrogenase in Cyanothece by monitoring the concentrations of dissolved H2 and O2 in the cell suspension using a mass spectrometer with a permeable membrane inlet.