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.     

Characterization of heme-binding properties of Paracoccus denitrificans Surf1 proteins

Biogenesis of cytochrome c oxidase (COX) is a highly complex process involving >30 chaperones in eukaryotes; those required for the incorporation of the copper and heme cofactors are also conserved in bacteria. Surf1, associated with heme a insertion and with Leigh syndrome if defective in humans, is present as two homologs in the soil bacterium Paracoccus denitrificans, Surf1c and Surf1q. In an in vitro interaction assay, the heme a transfer from purified heme a synthase, CtaA, to Surf1c was followed, and both Surf proteins were tested for their heme a binding properties. Mutation of four strictly conserved amino acid residues within the transmembrane part of each Surf1 protein confirmed their requirement for heme binding. Interestingly the mutation of a tryptophan residue in transmembrane helix II (W200 in Surf1c and W209 in Surf1q) led to a drastic switch in the heme composition, with Surf1 now being populated mostly by heme o, the intermediate in the heme a biosynthetic pathway. This tryptophan residue discriminates between the two heme moieties, apparently coordinates the formyl group of heme a, and most likely presents the cofactor in a spatial orientation suitable for optimal transfer to its target site within subunit I of cytochrome c oxidase.     

Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at -40 degrees C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.     

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.     

Cytochrome oxidase assembly in yeast requires the product of COX11, a homolog of the P. denitrificans protein encoded by ORF3.

The synthesis of cytochrome oxidase in Saccharomyces cerevisiae was recently shown to require a protein encoded by the nuclear gene COX10. This protein was found to be homologous to the putative protein product of the open reading frame ORF1 reported in one of the cytochrome oxidase operons of Paracoccus denitrificans. In the present study we demonstrate the existence in yeast of a second nuclear gene, COX11, whose encoded protein is homologous to another open reading frame (ORF3) present in the same operon of P. denitrificans. Mutations in COX11 elicit a deficiency in cytochrome oxidase. In this and in other respects cox11 and cox10 mutants have very similar phenotypes. An antibody has been obtained against the yeast COX11 protein. The antibody recognizes a 28 kd protein in yeast mitochondria, consistent with the size of the protein predicted from the sequence of COX11. The COX11 protein is tightly associated with the mitochondrial membrane but is not a component of purified cytochrome oxidase. An analysis of cytochrome oxidase subunits in wild type and in a cox11 mutant suggests that the COX11 protein is not required either for synthesis or transport of the subunit polypeptides into mitochondria. It seems more probable that COX11 protein exerts its effect at some terminal stage of enzyme synthesis, perhaps in directing assembly of the subunits.     

Role of Surf1 in heme recruitment for bacterial COX biogenesis

Biogenesis of the mitochondrial cytochrome c oxidase (COX) is a highly complex process involving subunits encoded both in the nuclear and the organellar genome; in addition, a large number of assembly factors participate in this process. The soil bacterium Paracoccus denitrificans is an interesting alternative model for the study of COX biogenesis events because the number of chaperones involved is restricted to an essential set acting in the metal centre formation of oxidase, and the high degree of sequence homology suggests the same basic mechanisms during early COX assembly. Over the last years, studies on the P. denitrificans Surf1 protein shed some light on this important assembly factor as a heme a binding protein associated with Leigh syndrome in humans. Here, we summarise our current knowledge about Surf1 and its role in heme a incorporation events during bacterial COX biogenesis. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Subunit III of cytochrome c oxidase is expressed in Paracoccus denitrificans

Polyclonal antibodies have been obtained against a synthetic dodecapeptide identical to the aminoacid sequence 120-131 DSPIKDGVWPPE (inferred from its DNA sequence) of Paracoccus denitrificans cytochrome c oxidase subunit III. The antibodies had a titer higher than 1:10000 when tested against the antigen. These antibodies have been used to produce immunological evidence that, despite the fact that subunit III is not isolated with cytochrome c oxidase, it exists in Paracoccus denitrificans lysates. The antibodies did not show reactivity with bovine heart cytochrome c oxidase either by ELISA or immunoblotting. It was also shown that the antibodies react with a single polypeptide present in Paracoccus denitrificans cell lysates, having an apparent molecular weight close to that of subunit III of bovine heart oxidase.     

Assembly of the cytochrome bo3 complex

An understanding of the mechanisms that govern the assembly of macromolecular protein complexes is fundamental for studying their function and regulation. With this in mind, we have determined the assembly pathway for the membrane-embedded cytochrome bo(3) of Escherichia coli. We show that there is a preferred order of assembly, where subunits III and IV assemble first, followed by subunit I and finally subunit II. We also show that cofactor insertion catalyses assembly. These findings provide novel insights into the biogenesis of this model membrane protein complex.     

Genetics of Paracoccus denitrificans

Abstract In bioenergetic research Paracoccus denitrificans has been used as an interesting model to elucidate the mechanisms of bacterial energy transduction. Genes for protein complexes of the respiratory chain and for proteins which are involved in periplasmic electron transport have been cloned and sequenced. Conjugational gene transfer has allowed the construction of site-specific mutant strains. Complementation experiments did not only open the field for site-directed mutagenesis and investigation of the structure/function relationship of the various electron-transport proteins, but also allowed first insights into processes like oxygen-dependent gene regulation or the assembly of electron-transport complexes. Also data will be presented that characterize two restriction-/modification systems, the codon usage and the promoter sequences of Paracoccus . Details will be given about the extrachromosomal localization of a duplicated cytochrome oxidase subunit I gene on one of the Paracoccus megaplasmids.     

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.     

COX10 codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for the synthesis of yeast cytochrome oxidase

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to pet complementation group G19 lack cytochrome oxidase activity and cytochromes a and a3. The enzyme deficiency is caused by recessive mutations in the nuclear gene COX10. Analyses of cytochrome oxidase subunits suggest that the product of COX10 provides an essential function at a posttranslational stage of enzyme assembly. The wild type COX10 gene has been cloned by transformation of a mutant from complementation group G19 with a yeast genomic library. Based on the nucleotide sequence of COX10, the primary translation product has an Mr of 52,000. The amino-terminal 190 residues constitute a hydrophilic domain while the carboxyl-terminal region is hydrophobic and has nine potential membrane-spanning segments. The sequence of the carboxyl-terminal hydrophobic region is homologous to an unidentified protein encoded by a reading frame (ORF1) located in one of the cytochrome oxidase operons of Paracoccus denitrificans. The two proteins share 24% identical residues and exhibit very similar hydrophobicity profiles. The bacterial homolog, however, lacks the hydrophilic amino-terminal region of the yeast protein.     

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 ba₃-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa₃-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 aa₃-type cytochrome c oxidase and the ba₃-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.     

Are there isoenzymes of cytochrome c oxidase in Paracoccus denitrificans?

We have used a gene replacement strategy to delete the previously isolated gene [(1987) EMBO J. 6, 2825-2833] for the cytochrome c oxidase subunit I from Paracoccus denitrificans. The resulting mutant was still able to synthesize active cytochrome c oxidase. This led us to look for another locus which could completely suppress the mutation. In this study we report the isolation of a second gene encoding subunit I. An open reading frame coding for cytochrome c 550 was found upstream from this gene. We suggest that there are isoenzymes of cytochrome c oxidase (cytochrome aa3) in this bacterium.     

The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species.

The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.     

Paracoccus denitrificans CcmG is a periplasmic protein–disulphide oxidoreductase required for c- and aa3-type cytochrome biogenesis; evidence for a reductase role in vivo

Cloning and sequencing of the Paracoccus denitrificans ccmG gene indicates that it codes for a periplasmic protein–disulphide oxidoreductase; the presence of the sequence Cys-Pro-Pro-Cys at the CcmG active site suggests that it may act in vivo to reduce disulphide bonds rather than to form them. A CcmG–PhoA fusion confirmed the periplasmic location. Disruption of the ccmG gene resulted in not only the expected phenotype of pleiotropic deficiency in c -type cytochromes, but also loss of spectroscopically detectable cytochrome aa ₃, cytochrome c oxidase and ascorbate/TMPD oxidase activities; there was also an enhanced sensitivity to growth inhibition by some component of rich media and by oxidized thiol compounds. Dithiothreitol promoted the growth of the ccmG mutant on rich media and substantially restored spectroscopically detectable cytochrome aa ₃ and cytochrome c oxidase activity, although it did not restore c -type cytochrome biogenesis. Assembly of the disulphide-bridged proteins methanol dehydrogenase and Escherichia coli alkaline phosphatase was unaffected in the ccmG mutant. It is proposed that P. denitrificans CcmG acts in vivo to reduce protein–disulphide bonds in certain protein substrates including c -type cytochrome polypeptides and/or polypeptides involved in c -type cytochrome biogenesis.     

An inducible periplasmic blue copper protein from Paracoccus denitrificans. Purification, properties, and physiological role

When grown on methylamine as a sole carbon source, Paracoccus denitrificans synthesizes a Type I blue copper protein which mediates electron transfer between methylamine dehydrogenase and cytochrome c. This blue copper protein does not serve as an electron acceptor for methanol dehydrogenase and is not synthesized by cells grown on methanol or succinate. The blue copper protein and methylamine dehydrogenase were localized in the periplasm of P. denitrificans, whereas formate dehydrogenase was cytoplasmic. The copper protein can be purified to high yield in a single step from the periplasmic subcellular fraction prepared from P. denitrificans. The purified protein contains a single 15,000-Da polypeptide chain and one copper atom/molecule and exhibits a pI of 4.8. The oxidized form of the protein absorbs strongly at 595 nm and weakly at 464 nm. The physical and physiological properties of this protein indicate that it is not an azurin, but representative of another class of blue copper proteins.     

New pathway of amine oxidation respiratory chain of Paracoccus denitrificans IFO 12442.

The physiological electron acceptor of quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans IFO 12442 was identified by biochemical and electrochemical methods. Of three types of heme c-containing proteins purified together with QH-AmDH from the periplasm of n-butylamine-grown cells, only constitutive cytochrome c-550 was reduced by the addition of QH-AmDH and n-butylamine. Reconstitution of the respiratory chain revealed that cytochrome c-550 mediates the electron transfer from QH-AmDH to the terminal oxidase. This is a new pathway of the amine oxidation respiratory chain of P. denitrificans.     

Calmodulin stimulation of adenylate cyclase of intestinal epithelium

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.     

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.