Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration. The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water. Electron transport through the enzyme is coupled to the generation of a protonmotive force. The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu. The subunits are all encoded by the cyo operon. Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family. Gene fusions have been used to define the topology of each of the gene products. Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively. The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme. Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced. These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases. Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups.     

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.

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.     

Demonstration by FTIR that the bo-type ubiquinol oxidase of Escherichia coli contains a heme-copper binuclear center similar to that in cytochrome c oxidase and that proper assembly of the binuclear center requires the cyoE gene product.

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)     

Determination of the ligands of the low spin heme of the cytochrome o ubiquinol oxidase complex using site-directed mutagenesis.

The cytochrome o complex of Escherichia coli is a ubiquinol oxidase which is the predominant respiratory terminal oxidase when the bacteria are grown under high oxygen tension. The amino acid sequences of three of the subunits of this quinol oxidase reveal a substantial relationship to the aa3-type cytochrome c oxidases. The two cytochrome components (b563.5 and o) and the single copper (CuB) present in the E. coli quinol oxidase appear to be equivalent to cytochrome a, cytochrome a3, and CuB of the aa3-type cytochrome c oxidases, respectively. These three prosthetic groups are all located within subunit I of the oxidase. Sequence alignments indicate only six totally conserved histidine residues among all known sequences of subunit I of the cytochrome c oxidases of various species plus the E. coli quinol oxidase. Site-directed mutagenesis has been used to change each of these totally conserved histidines with the presumption that two of these six must ligate to the low spin cytochrome center of the E. coli oxidase. The presence of the low spin cytochrome b563.5 component of the oxidase can be evaluated both by visible absorbance properties and by its EPR spectrum. The results unambiguously indicate that His-106 and His-421 are the ligands of the six-coordinate low spin cytochrome b563.5. Although the data are not definitive in making additional metal ligation assignments of the remaining four totally conserved histidines, a reasonable model is suggested for the structure of the catalytic core of the cytochrome o complex and, by extrapolation, of cytochrome c oxidase.     

The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o terminal oxidase complex is a component of the aerobic respiratory chain of Escherichia coli. This enzyme catalyzes the oxidation of ubiquinol-8 to ubiquinone-8 within the cytoplasmic membrane and the concomitant reduction of O2 to H2O. The hydropathy profiles of the deduced amino acid sequences suggest that all five of the gene products of the cyo operon contain multiple membrane-spanning helical segments. The goal of this work was to obtain experimental evidence for the topology of the five gene products in the cytoplasmic membrane by using the technique of gene fusions. A number of random gene fusions were generated in vitro encoding hybrid proteins in which the amino-terminal portion was provided by the subunit of interest and the carboxyl-terminal portion by one of two sensor proteins, alkaline phosphatase lacking its signal sequence or beta-galactosidase. Results obtained are self-consistent, and topological models are proposed for all of the five gene products encoded by the cyo operon. Based on the sequence similarities with subunits of the aa3-type cytochrome c oxidases, the experimental evidence obtained here can be used to infer topological models for the mitochondrial encoded subunits of the eukaryotic cytochrome c oxidases.     

Two variants of the assembly factor Surf1 target specific terminal oxidases in Paracoccus denitrificans

Biogenesis of cytochrome c oxidase (COX) relies on a large number of assembly proteins, one of them being Surf1. In humans, the loss of Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder. In the soil bacterium Paracoccus denitrificans, homologous genes specifying Surf1 have been identified and located in two operons of terminal oxidases: surf1q is the last gene of the qox operon (coding for a ba(3)-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa(3)-type cytochrome c oxidase). We introduced chromosomal single and double deletions for both surf1 genes, leading to significantly reduced oxidase activities in membrane. Our experiments on P. denitrificans surf1 single deletion strains show that both Surf1c and Surf1q are functional and act independently for the aa(3)-type cytochrome c oxidase and the ba(3)-type quinol oxidase, respectively. This is the first direct experimental evidence for the involvement of a Surf1 protein in the assembly of a quinol oxidase. Analyzing the heme content of purified cytochrome c oxidase, we conclude that Surf1, though not indispensable for oxidase assembly, is involved in an early step of cofactor insertion into subunit I.     

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.     

Differential effects of glutamate-286 mutations in the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo(3) ubiquinol oxidase from Escherichia coli

Both the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcO(aa3)) and the closely related bo(3)-type ubiquinol oxidase from Escherichia coli (EcQO(bo3)) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcO(aa3) result in long range conformational changes that influence the protein interactions with both heme a and heme a(3). Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcO(aa3) to ~0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a(3)-Cu(B) active site. In contrast, the E286C mutation of EcQO(bo3) exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcO(aa3), the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQO(bo3) is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa3-600 quinol oxidase.

Bacillus subtilis contains two aa3-type terminal oxidases (caa3-605 and aa3-600) catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H2O (Lauraeus, M., Haltia, T., Saraste, M., and Wikstr?m, M. (1991) Eur. J. Biochem. 197, 699-705). Previous studies characterized only the structural genes of caa3-605 oxidase. We isolated the genes coding for the four subunits of a B. subtilis terminal oxidase from a genomic DNA library. These genes, named qoxA to qoxD, are organized in an operon. Examination of the deduced amino acid sequence of Qox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases. In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B. subtilis caa3-605 cytochrome c oxidase. We produced, by in vitro mutagenesis, a mutation in the qox operon. From the phenotype of the mutant strain devoid of Qox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein. Although several terminal oxidases are found in B. subtilis, Qox oxidase (aa3-600) is predominant during the vegetative growth and its absence leads to important alterations of the phenotype of B. subtilis.     

Expression of cyoA and cyoB demonstrates that the CO-binding heme component of the Escherichia coli cytochrome o complex is in subunit I

The cytochrome o complex of the Escherichia coli aerobic respiratory chain is a ubiquinol oxidase. The enzyme consists of at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and contains two heme b prosthetic groups (b555 and b562) plus copper. The sequence of the cyo operon, encoding the subunits of the oxidase, reveals five open reading frames, cyoABCDE. This paper describes results obtained by expressing independently cyoA and cyoB in the absence of the other subunits of the complex. Polyclonal antibodies which react with subunits I and II of the purified oxidase demonstrate that cyoA and cyoB correspond to subunit II and subunit I, respectively, of the complex. These subunits are stably inserted into the membrane when expressed. Furthermore, expression of cyoB (subunit I) results in elevated heme levels in the membrane. Reduced-minus-oxidized spectra suggest that the cytochrome b555 component is present but that the cytochrome b562 component is not. This heme component is shown to bind to CO, as it does in the intact enzyme. Hence, subunit I alone is sufficient for the assembly of the stable CO-binding heme component of this oxidase.     

Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides.

It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga. This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations. Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum. The FTIR spectra indicate that the binuclear center of the R. sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase. Upon deletion of the ctaD gene, encoding subunit I of the aa3-type oxidase, substantial cytochrome c oxidase remains in the membranes of aerobically grown R. sphaeroides. This correlates with a second wild-type R. sphaeroides is grown photosynthetically, the chromatophore membranes lack the aa3-type oxidase but have this second heme-copper oxidase. Subunit I of the heme-copper oxidase superfamily contains the binuclear center. Amino acid sequence alignments show that this subunit is structurally very highly conserved among both eukaryotic and prokaryotic species. The polymerase chain reaction was used to show that the chromosome of R. sphaeroides contains at least one other gene that is a homolog of ctaD, the gene encoding subunit I of the aa3-type cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of heme and copper ligands in subunit I of the cytochrome bo complex in Escherichia coli.

The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli (Kita, K., Konishi, K., and Anraku, Y. (1984) J. Biol. Chem. 259, 3368-3374) and functions as a proton pump. It belongs to the heme-copper oxidase superfamily with the aa3-type cytochrome c oxidases in mitochondria and aerobic bacteria. In order to identify ligands of hemes and copper, we have substituted eight conserved histidines in subunit I by alanine and, in addition, His-106, -284, and -421 by glutamine and methionine. Western immunoblotting analysis showed that all the mutations do not affect the expression level of subunit I in the cytoplasmic membrane, indicating that these histidines are not crucial for its stability. A single copy expression vector carrying a single mutation at the invariant histidines, His-106, His-284, His-333, His-334, His-419, and His-421, of subunit I was unable to support the aerobic growth of a strain in which the chromosomal terminal oxidase genes (the cyo and cyd operons) have been deleted. The same mutations caused a complete loss of ubiquinol oxidase activity of the partially purified enzymes. Spectroscopic analysis of mutant oxidases in the cytoplasmic membrane revealed that substitutions of His-106 and -421 specifically eliminated a 563.5 nm peak of the low spin heme and that replacements of His-106, -284, and -419 reduced the extent of the CO-binding high spin heme. These spectroscopic properties of mutant oxidases were further confirmed with partially purified preparations. Atomic absorption analysis showed that substitutions of His-106, -333, -334, and -419 eliminated CuB almost completely. Based on these findings, we conclude that His-106 and -421 function as the axial ligands of the low spin heme and His-284 is a possible ligand of the high spin heme. His-333, -334, and -419 residues are attributed to the ligands of CuB. We present a helical wheel model of the redox center in subunit I, which consists of the membrane-spanning regions II, VI, VII, and X, and discuss the implications of the model.     

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.     

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.     

Optical and resonance Raman spectroscopy of the heme groups of the quinol-oxidizing cytochrome aa3 of Bacillus subtilis.

The cytochrome aa3-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water. It resembles the aa3-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CuA center and the cytochrome-c oxidizing activity of the mitochondrial enzyme. We have used optical and resonance Raman spectroscopies to study the B. subtilis oxidase in detail. The alpha-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa3-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa3-600. The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa3-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa3-type oxidases. Raman modes arise at 194 and 214 cm-1 in aa3-600, whereas a single band at about 214 cm-1 is assigned to the iron-histidine stretch for the other aa3-type oxidases. Possible explanations for the occurrence of these two modes are discussed. Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa3-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.     

Paracoccus denitrificans mutants deleted in the gene for subunit II of cytochrome c oxidase also lack subunit I.

As a prerequisite to site-directed mutagenesis on cytochrome c oxidase, two different mutants are constructed by inactivating the cta gene locus encoding subunits II and III (ctaC and ctaE) of the Paracoccus denitrificans oxidase. Either a short fragment encoding part of the putative copper binding site near the C terminus of subunit II, or a substantial fragment, comprising parts of the coding region for both subunits and all of the intervening three open reading frames, are removed and replaced by the kanamycin resistance gene. Each construct, ligated into a suicide vector, is mated into Paracoccus, and mutants originating from double homologous recombination events are selected. We observe complete loss of alpha-type heme and of oxidase subunits, as well as a substantial decrease in the cytochrome c oxidase activity. Upon complementation with the ctaC gene (plus various lengths of downstream sequence extending into the operon), subunit II gets expressed in all cases. Wild-type phenotype, however, is only restored with the whole operon. Using smaller fragments for complementation gives interesting clues on roles of the open reading frames for the assembly process of the oxidase complex; two of the open reading frame genes most likely code for two independent assembly factors. Since homologous genes have been described not only for other bacterial oxidases, but their gene products shown to participate also in the assembly of the yeast enzyme, they seem to constitute a group of evolutionary conserved proteins.     

Heme-copper oxidases and their electron donors in cyanobacterial respiratory electron transport

Cyanobacteria are the paradigmatic organisms of oxygenic (plant-type) photosynthesis and aerobic respiration. Since there is still an amazing lack of knowledge on the role and mechanism of their respiratory electron transport, we have critically analyzed all fully or partially sequenced genomes for heme-copper oxidases and their (putative) electron donors cytochrome c(6), plastocyanin, and cytochrome c(M). Well-known structure-function relationships of the two branches of heme-copper oxidases, namely cytochrome c (aa(3)-type) oxidase (COX) and quinol (bo-type) oxidase (QOX), formed the base for a critical inspection of genes and ORFs found in cyanobacterial genomes. It is demonstrated that at least one operon encoding subunits I-III of COX is found in all cyanobacteria, whereas many non-N(2)-fixing species lack QOX. Sequence analysis suggests that both cyanobacterial terminal oxidases should be capable of both the four-electron reduction of dioxygen and proton pumping. All diazotrophic organisms have at least one operon that encodes QOX. In addition, the highly refined specialization in heterocyst forming Nostocales is reflected by the presence of two paralogs encoding COX. The majority of cyanobacterial genomes contain one gene or ORF for plastocyanin and cytochrome c(M), whereas 1-4 paralogs for cytochrome c(6) were found. These findings are discussed with respect to published data about the role of respiration in wild-type and mutated cyanobacterial strains in normal metabolism, stress adaptation, and nitrogen fixation. A model of the branched electron-transport pathways downstream of plastoquinol in cyanobacteria is presented.