Paracoccus denitrificans cytochrome c1 gene replacement mutants.

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.     

Preparation and characterization of the water-soluble heme-binding domain of cytochrome c1 from the Rhodobacter sphaeroides bc1 complex.

The ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides consists of four subunits. One of these subunits, cytochrome c1, is the site of interaction with cytochrome c2, a periplasmic protein. In addition, the sequences of the fbcC gene and of the cytochrome c1 subunit that it encodes suggest that the protein should be located on the periplasmic side of the cytoplasmic membrane and that it is anchored to the membrane by a single membrane-spanning alpha-helix located at the carboxyl-terminal end of the polypeptide. Site-directed mutagenesis of the fbcC gene was used to alter the codon for Gln228 to a stop codon. This results in the production of a truncated version of the cytochrome c1 subunit that lacks the membrane anchor at the carboxyl terminus. The bc1 complex fails to assemble properly as a result of this mutation, but the Rb. sphaeroides cells expressing the altered gene contain a water-soluble form of cytochrome c1 in the periplasm. The water-soluble cytochrome c1 was purified and characterized. The amino-terminal sequence is identical with that of the membrane-bound subunit, indicating the signal sequence is properly processed. High pressure liquid chromatography gel filtration chromatography indicates it is monomeric (28 kDa). The heme content and electrochemical properties are similar to those of the intact subunit within the complex. Flash-induced electron transfer kinetics measured using whole cells demonstrated that the water-soluble cytochrome c1 is competent as a reductant for cytochrome c2 within the periplasmic space. These data show that the isolated water-soluble cytochrome c1 retains many of the properties of the membrane-bound subunit of the bc1 complex and, therefore, will be useful for further structural and functional characterization.     

Large-scale purification and characterization of a highly active four-subunit cytochrome bc1 complex from Rhodobacter sphaeroides

A highly active, large-scale preparation of ubiquinol:cytochrome c2 oxidoreductase (EC 1.10.2.2; cytochrome bc1 complex) has been obtained from Rhodobacter sphaeroides. The enzyme was solubilized from chromatophores by using dodecyl maltoside in the presence of glycerol and was purified by anion-exchange and gel filtration chromatography. The procedure yields 35 mg of pure bc1 complex from 4.5 g of membrane protein, and its consistently results in an enzyme preparation that catalyzes the reduction of horse heart cytochrome c with a turnover of 250-350 (mumol of cyt c reduced).(mumol of cyt c1)-1.s-1. The turnover number is at least double that of the best preparation reported in the literature [Ljungdahl, P. O., Pennoyer, J. D., Robertson, D. C., & Trumpower, B. L. (1987) Biochim. Biophys. Acta 891, 227-241]. The scale is increased 25-fold, and the yield is markedly improved by using this protocol. Four polypeptide subunits were observed by SDS-PAGE, with Mr values of 40K, 34K, 24K, and 14K. N-Terminal amino acid sequences were obtained for cytochrome c1, the iron-sulfur protein subunit, and for cytochrome b and were identical with the expected protein sequences deduced from the DNA sequence of the fbc operon, with the exceptions that a 22-residue fragment is processed off of the N-terminus of cytochrome c1 and the N-terminal methionine residue is cleaved off both the b cytochrome and iron-sulfur protein subunits. Western blotting experiments indicate that subunit IV is not a contaminating light-harvesting complex polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides. Characterization of fbc deletion mutants and complementation by a site-specific mutational variant

The ubiquinol: cytochrome-c oxidoreductase (cytochrome bc1 complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc1 complex has a dual function. When the cells are grown photosynthetically, the bc1 complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc1 complex is a necessary component of the cytochrome-c2-dependent respiratory chain. The fact that the bc1 complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site-directed mutagenesis to address questions relating to the structure and function of the bc1 complex. In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe-2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc1 complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc1 complex which, as expected, is resistant to the inhibitor. Hence, this system is suitable for the detailed characterization of the bc1 complex, combining site-directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.     

The genes of the Paracoccus denitrificans bc1 complex. Nucleotide sequence and homologies between bacterial and mitochondrial subunits

The genes for the three subunits of the cytochrome bc1 complex from the bacterium Paracoccus denitrificans were identified by screening a gene library constructed in pBR 322 for expression using a cytochrome c1-specific antibody. These three genes coding for the FeS subunit, cytochrome b, and cytochrome c1 were located on contiguous sites on the genome in a presumed operon arrangement. The DNA-deduced amino acid sequence shows that all three subunits are homologous to corresponding polypeptides of the mitochondrial cytochrome bc1 complex. Cytochrome c1 of Paracoccus is much larger than its mitochondrial counterpart due to an extra 150 amino acids of unique, highly acidic composition; in addition, it is most likely synthesized as a precursor polypeptide.     

fbc operon, encoding the Rieske Fe-S protein cytochrome b, and cytochrome c1 apoproteins previously described from Rhodopseudomonas sphaeroides, is from Rhodopseudomonas capsulata

Detailed comparison of the 'Rhodopseudomonas sphaeroides GA' strain used by Gabellini et al. (1985) with genuine R. sphaeroides and R. capsulata strains indicated that the previously reported fbc operon of R. sphaeroides (Gabellini and Sebald, 1986) encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 subunits of the ubiquinol:cytochrome c2 oxidoreductase, is not from R. sphaeroides, but is rather from a strain of R. capsulata. Consequently, the genuine bc1 genes from R. sphaeroides were cloned using corresponding R. capsulata genes as probes, and a partial nucleotide sequence for the Rieske Fe-S protein of R. sphaeroides was determined and compared with that of R. capsulata.     

Primary structure of the bc1 complex of Rhodopseudomonas capsulata. Nucleotide sequence of the pet operon encoding the Rieske cytochrome b, and cytochrome c1 apoproteins

The nucleotide sequence of the pet operon of Rhodopseudomonas capsulata strain SB1003 has been determined. This operon consists of the petA, petB and petC genes, which encode the Rieske Fe-S protein, cytochrome b and cytochrome c1, respectively, all components of the ubiquinol-cytochrome c2 oxidoreductase. The deduced amino acid sequences of the pet genes show homology to the corresponding proteins from other organisms, and particularly high homologies (over 90% for amino acid and nucleotide sequences) to the previously described fbc operon from a strain previously identified as Rhodopseudomonas spheroides GA. The amino acid sequences of the pet proteins are discussed with reference to the structure and function of the ubiquinol-cytochrome c2 oxidoreductase.     

An Engineered Cytochrome b6c1 Complex with a Split Cytochrome b Is Able To Support Photosynthetic Growth of Rhodobacter capsulatus

The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.     

Organization of the genes coding for the reaction-centre L and M subunits and B870 antenna polypeptides α and β from the aerobic photosynthetic bacterium Erythrobacter species OCH114

In the aerobic photosynthetic bacterium Erythrobacter species OCH114 the structural genes coding for the light-harvesting (LH) complex B870 and the reaction-centre (RC) polypeptides (the gene products of the pufB, pufA, pufL and pufM genes) are mapped on a 2.728 kbp EcoRI fragment. Sequencing of this fragment revealed that the deduced amino acid sequences contain 50 (B870 beta), 52 (B850 alpha), 283 (RCL) and 331 (RCM) residues with the corresponding molecular weights of 5592, 5814, 31364, and 37671, respectively. In the corresponding mRNA a 'hairpin' structure (delta G degrees = -26.6 kcal) is predicted to be located immediately downstream of pufA. The RC and LH polypeptides are highly homologous to those of the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis. Directly downstream of pufM there is an open reading frame (ORF) of unknown size. Partial sequencing indicates that this ORF is highly homologous to the cytochrome subunit of the photosynthetic reaction centre from R. viridis. In the puf operon no pufQ or pufX genes could be found, but the bchA gene is located upstream of that operon. Plasmid pESS8.9 containing the 2.728 kbp EcoRI fragment reconstituted a photoinactive mutant of Erythrobacter species OCH114. Comparative analysis of the DNA region upstream of the puf operon and of bacteriochlorophyll (Bchl) synthesis indicated that Bchl synthesis and puf gene expression are regulated differently in Erythrobacter and purple bacteria, respectively.     

Rhodobacter capsulatus mutants lacking the Rieske FeS protein form a stable cytochrome bc1 subcomplex with an intact quinone reduction site.

The ubiquinol-cytochrome c oxidoreductase (or bc1 complex) of Rhodobacter capsulatus consists of three subunits: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, encoded by the fbcF, fbcB, and fbcC genes, respectively. In the preceding paper [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry (preceding paper in this issue)], we have observed that the apoproteins for cytochromes b and c1 are fully present in the intracytoplasmic membrane of R. capsulatus mutants containing low amounts of, or no, Rieske apoprotein. Here we present evidence that the redox midpoint potentials of cytochromes b and c1, as well as their ability to bind antimycin and stabilize a semiquinone at the Qi site, are unaffected by the absence of the Rieske subunit. This is the first report describing a mutant containing a stable bc1 subcomplex with an intact Qi site in the chromatophore membranes, and provides further evidence that a functional quinone reduction site can be formed in the absence of a quinol oxidation (Qo) site. Additional mutants carrying fbc deletions expressing the remaining subunits of the cytochrome bc1 complex were constructed to investigate the relationship among these subunits for their stability in vivo. Western blot analysis of these mutants indicated that cytochromes b and c1 protect each other against degradation, suggesting that they form a two-protein subcomplex in the absence of the Rieske protein subunit.     

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.     

Nucleotide sequence of the gene coding for four subunits of cytochrome c oxidase from the thermophilic bacterium PS3

The gene coding for four subunits of cytochrome aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/caaB product), 333 residues for subunit II (CO2/caaA product), 207 residues for subunit III (CO3/caaC product), and 109 residues for subunit IV (CO4/caaD product) after processing. Re-examination of the sequencing of caa revealed a longer open reading frame for CO1, which contains 14 transmembrane segments instead of 12 [Sone et al. (1988) J. Biochem. 103, 606-610], although the main portions of the sequences constituting cytochrome a (FeA), cytochrome a3 (FeB), and CuB are correct. PS3 CO2 has an additional sequence for cytochrome c after the CuA binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart. PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondrial counterpart, which has 7 segments. The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome bo-type oxidase from Escherichia coli as well, in spite of the difference of hemes. PS3 CO3 and CO4 are much more similar to E. coli CO3 and CO4 than to mitochondrial CO3 and CO4, respectively.     

Nucleotide sequence of the gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex.

The nucleotide sequence was determined for the first part of the Bacillus subtilis sdh operon. An open reading frame corresponding to the structural gene, sdhA, for cytochrome b558 was identified. The predicted molecular weight of the cytochrome (excluding the N-terminal methionine) is 22,770. It is a very hydrophobic protein with five probable membrane-spanning segments. There is little homology between the B. subtilis cytochrome b558 and cytochrome b of mitochondrial complex III from different organisms or between cytochrome b558 and the hydrophobic sdhC and sdhD peptides of the Escherichia coli sdh operon. About 30 bases downstream of the sdhA stop codon, a new open reading frame starts. The nucleotide sequence predicts the presence of a typical flavin-binding peptide which identifies this reading frame as part of the sdhB gene. Seven bases upstream of the sdhA initiation codon ATG there is a typical B. subtilis ribosome binding site (free energy of interaction, -63 kJ), and further upstream, tentative sigma 55 and sigma 32 promoter sequences were found. The upstream region also contains two 12-base-long direct repeats; their significance is unknown.     

Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase.

The gene ald, encoding aldehyde dehydrogenase, has been cloned from a genomic library of Escherichia coli K-12 constructed with plasmid pBR322 by complementing an aldehyde dehydrogenase-deficient mutant. The ald region was sequenced, and a single open reading frame of 479 codons specifying the subunit of the aldehyde dehydrogenase enzyme complex was identified. Determination of the N-terminal amino acid sequence of the enzyme protein unambiguously established the identity and the start codon of the ald gene. Analysis of the 5'- and 3'-flanking sequences indicated that the ald gene is an operon. The deduced amino acid sequence of the ald gene displayed homology with sequences of several aldehyde dehydrogenases of eukaryotic origin but not with microbial glyceraldehyde-3-phosphate dehydrogenase.     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast.

The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.     

Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.     

The primary structure of cytochrome c from the nematode Caenorhabditis elegans.

The complete amino acid sequence of cytochrome c from the nematode Caenorhabditis elegans was determined. The native protein displays the same spectral properties in the oxidized and reduced states as horse heart cytochrome c. The apoprotein consists of 110 amino acid residues and differs from human cytochrome c by 44 substitutions, one internal deletion, five N-terminal additions and two C-terminal additions. One of the substitutions is the replacement of an 'invariant' phenylalanine residue at position 15 by tyrosine. The N-terminal sequence extension contains a short peptide motif, which is highly homologous with a peptide fragment present at the N-terminus of annelid and insect cytochrome c sequences. From the number of amino acid changes and the evolutionary rate of cytochrome c it would appear that nematodes diverged from a line leading to man about 1.4 billion years ago. When similar data based on the amino acid sequences of the histones H1, H2A, H2B and H3 are taken into account, the average estimate is 1.1 +/- 0.1 billion years.