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.     

Deletion of the 6-kDa subunit affects the activity and yield of the bc1 complex from Rhodovulum sulfidophilum.

The cytochrome bc1 complex from Rhodovulum sulfidophilum purifies as a four-subunit complex: the cytochrome b, cytochrome c1 and Rieske iron-sulphur proteins, which are encoded together in the fbc operon, as well as a 6-kDa protein. The gene encoding the 6-kDa protein, named fbcS, has been identified. It is located within the sox operon, which encodes the subunits of sarcosine oxidase. The encoded 6-kDa protein is very hydrophobic and is predicted to form a single transmembrane helix. It shows no sequence homology to any known protein. The gene has been knocked-out of the genome and a three-subunit complex can be purified. This deletion leads to a large reduction in the yield of the isolated complex and in its activity compared to wild-type. The high quinone content found in the wild-type complex is, however, maintained after removal of the 6-kDa protein. Surprisingly, a fourth subunit of approximately 6 kDa is again found to copurify with the Rhv. sulfidophilum bc1 complex when only the fbc operon is expressed heterologously in a near-relative, Rhodobacter capsulatus, which lacks this small subunit in its own bc1 complex.     

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.     

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)     

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, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides

The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase.     

Biogenesis of the yeast cytochrome bc1 complex

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast 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.     

The extra fragment of the iron-sulfur protein (residues 96-107) of Rhodobacter sphaeroides cytochrome bc1 complex is required for protein stability

Sequence alignment of the Rieske iron-sulfur protein (ISP) of cytochrome bc(1) complex from various sources reveals that bacterial ISPs contain an extra fragment. To study the role of this fragment in bacterial cytochrome bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with deletion or single- or multiple-alanine substitution at various positions of this fragment (residues 96-107) were generated and characterized. The ISPDelta(96-107), ISP(96-107)A, and ISP(104-107)A mutant cells, in which residues 96-107 of ISP are deleted, and residues 96-107 and 104-107 are substituted with alanine, respectively, do not grow photosynthetically and show no bc(1) complex activity in intracytoplasmic membranes prepared from these mutant cells. The ISP(96-99)A, in which residues 96-99 are substituted with alanine, grows photosynthetically at a rate comparable to that of the complement cells, whereas ISP(100-103)A, in which residues 100-103 are substituted with alanine, has a longer lag period prior to photosynthetic growth. Chromatophores prepared from these two mutant cells have 48% and 9% of the bc(1) activity found in the complement chromatophores. The loss (or decrease) of bc(1) activity in these mutant membranes results from a lack (or decrease) of ISP in the membrane due to ISP protein instability and not from mutations affecting the assembly of cytochromes b and c(1) into the membrane, the binding affinity of cytochrome b to cytochrome c(1), or the ability of these two cytochromes to interact with ISP or subunit IV. The order of essentiality of residues in this fragment is residues 104-107 > residues 100-103 > residues 96-99.     

Deletion of subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex specifically impairs electron transfer at the ubiquinol oxidase site (center P) in the bc1 complex.

Deletion of QCR9, the nuclear gene encoding subunit 9 of the mitochondrial cytochrome bc1 complex in Saccharomyces cerevisiae, results in inactivation of the bc1 complex and inability of the yeast to grow on non-fermentable carbon sources. The loss of bc1 complex activity is due to loss of electron transfer activity at the ubiquinol oxidase site (center P) in the complex. Electron transfer at the ubiquinone reductase site (center N), is unaffected by the loss of subunit 9, but the extent of cytochrome b reduction is diminished. This is the first instance in which a supernumerary polypeptide, lacking a redox prosthetic group, has been shown to be required for an electron transfer reaction within the cytochrome bc1 complex.     

Uncovering the molecular mode of action of the antimalarial drug atovaquone using a bacterial system

Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c1 of the cytochrome bc1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc1 complex.     

The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.     

A four-subunit cytochrome bc(1) complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc(1) complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc(1) subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc(1) complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c(1) carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c(552), mediating electron transfer to the ba(3) oxidase. Identification of this cytochrome bc(1) complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.     

Allosteric properties of cyanobacterial cytochrome c oxidase: inhibition of the coupled enzyme by ATP and stimulation by ADP

Thorough analysis of the cta operon of Synechocystis sp. PCC6803 (grown in high-concentration salt medium to enhance the expression of respiratory proteins) showed that, apart from ctaCDE and Fb genes potentially encoding subunits I, II, III, and a small pseudo-bacteria-like subunit-IV of unknown function, a large mitochondria-like cta-Fm gene and a pronounced terminator structure are additional components of the operon. The deduced cta Fm gene product shows approximately 50% and 20% sequence identity to the Saccharomyces cerevisiae and beef heart mitochondrial COIV proteins, respectively. It also shows amino acid regions (near the N terminus, on the cytosolic side) with conspicuous sequence similarities to adenylate-binding proteins such as ATP synthase beta subunit Walker A and B consensus regions or to adenylate kinase. We suggest that, similar to the situation with beef heart mitochondria, it is the mitochondria-like subunit-IV of the cyanobacterial aa3-type cytochrome-c oxidase that confers allosteric properties to the cyanobacterial enzyme, the H+/e- ratios of cytochrome c oxidation being significantly lowered by ATP (intravesicular or intraliposomal) but enhanced by ADP. Therefore, the antagonistic action of ATP and ADP was in a way that the redox reaction proper, was always significantly less affected than the coupled proton translocation. Evolutionary and ecological implications of the unusual allosteric regulation of a prokaryotic cytochrome-c oxidase is discussed.     

petR, located upstream of the fbcFBC operon encoding the cytochrome bc1 complex, is homologous to bacterial response regulators and necessary for photosynthetic and respiratory growth of Rhodobacter capsulatus

Interposon mutagenesis of a region upstream of the petABC(fbcFBC) operon, encoding the ubiquinol: cytochrome c2 oxidoreductase (bc1 complex) of the photosynthetic bacterium Rhodobacter capsulatus revealed the presence of two genes, petP and petR. DNA nucleotide sequence determination of this region indicated that petP and petR are transcribed in the same direction as the petABC(fbcFBC) operon, and are translationally coupled. A silent insertion located in the interoperonal region separating petPR and the petABC(fbcFBC) genes indicated that these clusters have separate promoters. The deduced amino acid sequence of the putative petR gene product is homologous to various bacterial response regulators, especially to those of the OmpR subgroup. Moreover, it was found that PetR mutants are unable to grow on rich or minimal media by either photosynthesis or respiration, demonstrating that these gene products are essential for growth of R. capsulatus.     

The pet operon,encoding the prosthetic group-containing subunits of the cytochrome bc1 complex,of the purple sulfur bacterium Chromatium vinosum

The pet operon, encoding the prosthetic group-containing subunits of the cytochrome bc ₁ complex of the purple sulfur bacterium Chromatium vinosum, has been cloned and sequenced. The 5 to 3 order of the C. vinosum genes is: petA, encoding the Rieske iron-sulfur protein; petB, encoding cytochrome b; and petC, encoding cytochrome c₁. Cytochrome b is the best conserved subunit of the C. vinosum complex, when compared to the corresponding proteins from four photosynthetic purple non-sulfur bacteria (70 to 74% identity). Identities for the C. vinosum Rieske protein and those from purple non-sulfur bacteria range from 60 to 64%. The C-terminal region of the C. vinosum Rieske protein is quite similar to those of purple non-sulfur bacteria, while the N-terminal region is more closely related to mitochondrial Rieske proteins of organisms such as Neurospora crassa. Cytochrome c₁ is the least well-conserved protein of the C. vinosum cytochrome bc₁ complex, with identities ranging from 49 to 51% when compared to the corresponding proteins from purple non-sulfur bacteria. A well-conserved negatively-charged region of the cytochromes c₁ of the purple non-sulfur bacteria, thought to be involved in binding the electron acceptor for the complex, cytochrome c₂, is absent in C. vinosum cytochrome c₁. A positive Southern hybridization using a probe constructed from the Rhodobacter sphaeroides fbcQ gene, which codes for a fourth subunit of the cytochrome bc₁ complex in that bacterium, suggests the presence of a homologous gene in C. vinosum.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.