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.     

Purification and properties of Paracoccus denitrificans azurin

The isolation of an azurin type Cu protein from Paracoccus denitrificans (ATCC 13543) is described and some properties are reported. The purified protein has a molecular weight of 13,790 in a single polypeptide chain and contains one Cu atom per molecule. Its spectrum is typical of Type I, “blue” Cu proteins in showing an intense band at 595 nm; but it also shows a weaker absorption band at 448 nm. Its standard reduction potential has been measured to be +230 mV, which is the lowest potential observed to date for azurins isolated from bacterial sources. The purified protein shows fivefold greater electron transport activity with membrane fragments than with the soluble nitrite reductase of Paracoccus. This argues against the latter as the primary physiological oxidase system for azurin.     

Cytochrome c1 from Paracoccus denitrificans

Cytochrome c1 was purified from the bacterium Paracoccus denitrificans. It is an acidic, hydrophobic polypeptide with an apparent molecular weight of around 65000 and a single, covalently attached heme; it cross-reacts immunologically with cytochrome c1 from yeast mitochondria. The amino acid sequence of the tryptic heme peptide of the bacterial cytochrome c1 shows extensive homology to the corresponding region of beef heart cytochrome c1 [Wakabayashi, S. et al. (1982) J. Biol. Chem. 257, 9335-9344]. Positive evidence for a stable association of the Paracoccus cytochrome c1 with other polypeptides and b-type heme components ('bc1-complex') has not yet been obtained.     

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.     

Redox properties of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Paracoccus denitrificans produces two primary enzymes for the amine oxidation, tryptophan-tryptophylquinone (TTQ)-containing methylamine dehydrogenase (MADH) and quinohemoprotein amine dehydrogenase (QH-AmDH). QH-AmDH has a novel cofactor, cysteine tryptophylquinone (CTQ) and two hemes c. In this work, the redox potentials of three redox centers in QH-AmDH were determined by a mediator-assisted continuous-flow column electrolytic spectroelectrochemical technique. Kinetics of the electron transfer from QH-AmDH to three kinds of metalloproteins, amicyanin, cytochrome c(550), and horse heart cytochrome c were examined on the basis of the theory of mediated-bioelectrocatalysis. All these metalloproteins work as a good electron acceptor of QH-AmDH and donate the electron to the terminal oxidase of P. denitrificans, which was revealed by reconstitution of the respiratory chain. These properties are in marked contrast with those of MADH, which shows high specificity to amicyanin. These electron transfer kinetics are discussed in terms of thermodynamics and structural property.     

Outer membrane proteins induced upon iron deprivation of Paracoccus denitrificans

Deprivation of two strains of Paracoccus denitrificans of iron elicited substantial siderophore production, and the associated cell envelope protein changes were examined. In strain DSM 413 four new outer membrane proteins in the M_r range 84000 to 76000 were induced, and an M_r 23000 protein disappeared, upon iron deprivation. In strain DSM 65 three new outer membrane proteins in the M_r range 81000 to 76000 were induced.     

Purification and properties of thiamine pyrophosphokinase in Paracoccus denitrificans

The existence of thiamine pyrophosphokinase [EC 2.7.6.2] in procaryotic cells was first demonstrated in Paracoccus denitrificans (J. Bacteriol, (1976) 126, 1030-1036). The enzyme was therefore purified from this organism to determine its molecular structure and properties. Thiamine pyrophosphokinase which was purified 620-fold from P. denitrificans showed a single band on both polyacrylamide and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular weight in the latter case was calculated to be 23,000. Gel filtration analysis using Sephadex G-150 gave a molecular weight of 44,000, indicating that this enzyme contains at least two identical subunits. Although sedimentation equilibrium analysis gave a molecular weight of 96,000, indirect evidence suggests that the form having this molecular weight is an aggregate of the functional dimer. The activity of the purified enzyme required thiamine, ATP, and Mg2+, and the enzyme catalyzed thepyrophosphorylation of thiamine by ATP. Km values for thiamine and ATP were 10 microM and 0.38 mM, respectively. The activity was competitively inhibited by pyrithiamine, giving a Ki value of 19 microM. Oxythiamine and chloroethylthiamine were very weak inhibitors of the enzyme. The activity was also inhibited by the product, TPP.     

Purification and some properties of isocitrate dehydrogenase from Paracoccus denitrificans

NADP-dependent isocitrate dehydrogenase (ICDH) from the bacterium Paracoccus denitrificans was purified to homogeneity. The purification procedure involved ammonium sulphate fractionation, ion exchange chromatography, and gel permeation chromatography. The specific activity of purified ICDH was 801 nkat/mg, the yield of the enzyme 58%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. ICDH is a dimer composed of two probably identical subunits of relative molecular weight 90,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 5.6; the presence of Mn2+ is essential for enzyme activity. The absorption and fluorescence spectra of the homogeneous enzyme were measured as well.     

Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans

Highly active succinate-ubiquinone reductase has been purified from cytoplasmic membranes of aerobically grown Paracoccus denitrificans. The purified enzyme has a specific activity of 100 units per mg protein, and a turnover number of 305 s-1. Succinate-ubiquinone reductase activity of the purified enzyme is inhibited by 3'-methylcarboxin and thenoyltrifluoroacetone. Four subunits, with apparent molecular masses of 64.9, 28.9, 13.4 and 12.5 kDa, were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains 5.62 nmol covalently bound flavin and 3.79 nmol cytochrome b per mg protein. The 64.9 kDa subunit was shown to be a flavoprotein by its fluorescence. Polyclonal antibodies raised against this protein cross-reacted with the flavoprotein subunit of bovine heart mitochondrial succinate-ubiquinone reductase. The 28.9 kDa subunit is likely analogous to the bovine heart iron protein, and the cytochrome b heme is probably associated with one or both of the low-molecular-weight polypeptides. The cytochrome b is not reducible with succinate but is reoxidized with fumarate after prereduction with dithionite. Iron-sulfur clusters S-1 and S-3 of the Paracoccus oxidoreductase exhibit EPR spectra very similar to their mitochondrial counterparts. Paracoccus succinate-ubiquinone reductase complex is thus similar to the bovine heart mitochondrial enzyme with respect to prosthetic groups, enzymatic activity, inhibitor sensitivities, and polypeptide subunit composition.     

Properties of Paracoccus denitrificans amicyanin

Paracoccus denitrificans synthesizes an inducible, periplasmic, blue copper protein [Husain, M., & Davidson, V.L. (1985) J. Biol. Chem. 260, 14626-14629] that can be classified as an amicyanin on the basis of its ability to accept electrons from methylamine dehydrogenase. The amino acid composition and sequence of the 10 N-terminal residues of this protein have been determined. From these data, it is evident that amicyanin is structurally distinct from azurins as it contains no disulfide bond and an N-terminal sequence that is completely different from the highly conserved N-terminal azurin sequences. Dialysis of reduced amicyanin against potassium cyanide resulted in a nearly quantitative yield of apoamicyanin. Amicyanin and apoamicyanin exhibit fluorescence emission maxima at 314 nm when excited at 280 nm. Addition of 6 M guanidine hydrochloride shifts these emission maxima to 350 nm. The fluorescence intensity of apoamicyanin is 10-fold greater than that of amicyanin. Addition of copper to the apoprotein caused a stoichiometric quenching of fluorescence and restoration of visible absorbance with no concomitant change in absorbance at 280 nm. At least one cysteine residue, which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) in apoamicyanin, does not react in the holoprotein, even in the presence of 6 M guanidine hydrochloride. Reductive and oxidative titrations of amicyanin indicate that it is a one-electron carrier. This amicyanin is also able to accept electrons from the methylamine dehydrogenase isolated from bacterium W3A1, which is taxonomically very different from P. denitrificans.     

Spectral and electrochemical properties of glutaryl-CoA dehydrogenase from Paracoccus denitrificans

Studies of the spectral (UV/vis and resonance Raman) and electrochemical properties of the FAD-containing enzyme glutaryl-CoA dehydrogenase (GCD) from Paracoccus denitrificans reveal that the properties of the oxidized enzyme (GCDox) appear to be invariant from those properties known for other acyl-CoA dehydrogenases such as mammalian general acyl-CoA dehydrogenase (GACD) and butyryl-CoA dehydrogenase (BCD) from Megasphaera elsdenii. However, when either free or complexed GCD is reduced, its spectral and electrochemical behavior differs from that of both GACD and BCD. Free GCD does not stabilize any form of one-electron-reduced GCD, but when GCD is complexed to its inhibitor, aceto-acetyl-CoA, the enzyme stabilizes 20% of the blue neutral radical form of FAD (FADH.) upon reduction. Like GACD, when crotonyl-CoA- (CCoA) bound GCD is reduced, the red anionic form of FAD radical (FAD.-) is stabilized, and excess reduction equivalents are necessary to effect full reduction of the complex. A comproportionation reaction is proposed between fully reduced crotonyl-CoA-bound GCD (GCD2e-CCoA) and GCDox-CCoA to partially explain the stabilization of GCD-bound FAD.- by CCoA. When GCD is reduced by its optimal substrate, glutaryl-CoA, a two-electron reduction is observed with concomitant formation of a long-wavelength charge-transfer band. It is proposed that the ETF specific for GCD abstracts one electron from this charge-transfer species and this is followed by the decarboxylation of the oxidized substrate. At pH 6.4, potential values measured for free GCD and GCD bound to acetoacetyl-CoA are -0.085 and -0.129 V, respectively. Experimental evidence is given for a positive shift in the reduction potential of GCD when the enzyme is bound to a 1:1 mixture of butyryl-CoA and CCoA. However, significant GCD hydratase activity is observed, preventing quantitation of the potential shift.     

Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans.

Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells. The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits. Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group. The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources. The enzyme was able to catalyze the oxidation of a wide variety of primary aliphatic amines and diamines, but it did not react with secondary, tertiary, or aromatic amines. The enzyme exhibited optimal activity at pH 7.5, with Km values of 12.5 microM for methylamine and 156 microM for phenazine ethosulfate and a Vmax of 16.9 mumol/min per mg of protein. No loss of enzyme activity was observed after incubation for 48 h at pH values ranging from 3.0 to 10.5, and the enzyme was very stable to thermal denaturation. Enzyme activity and immunological detection of each subunit were only observed with cells which had been grown on methylamine as a carbon source.     

ATP-AMP phosphotransferase from Paracoccus denitrificans

1H NMR is used to study the solution structure of vitamin-D-induced bovine intestinal calcium-binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein folds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially. Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80 s-1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein.     

Characterization of a Paracoccus denitrificans mutant pleiotropically impaired in nitrogen metabolism

A Tn5 insertional prototrophic mutant of Paracoccus denitrificans (UBM219) was generated which grew on high (>1 mM) but not low (<0.5 mM) ammonium as sole nitrogen source. It did not utilize nitrate and most amino acids except glutamate and aspartate. UBM219 showed more than 10-fold lower levels of ammonium (methylammonium) transport, aspartate and alanine aminotransferase, but more than 10-fold higher activities of glutamate dehydrogenase and glutamate synthase. This pleiotropy indicates a mutation in a regulatory gene affecting nitrogen metabolism in general. — Ammonia assimilation pathways and regulation in Paracoccus resemble the patterns in enterobacteria with the exception, that alanine is generated by amino transfer from glutamate to pyruvate.Non-standard abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GluDH glutamate dehydrogenase - GPT glutamate/pyruvate aminotransferase - GOT glutamate/oxaloacetate aminotransferase     

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.     

Iron-regulated outer membrane proteins and non-siderophore-mediated iron acquisition by Paracoccus denitrificans

Abstract Deprivation of Paracoccus denitrificans of iron in sodium molybdate-containing medium caused a slower rate of growth and lower final cell yield, in contrast to our previous studies in non-sodium molybdate-containing medium, where iron deprivation had little effect on growth rate. Five high M _r outer membrane proteins and catechol production were induced in iron-deprived cultures. The fifth protein, M _r 72 000, was produced later than the others. Growth of iron-deprived cells in medium containing 20 μM ferric citrate repressed siderophore and iron deprivation-induced protein production, and led to production of an M _r 23 000 outer membrane protein (half maximum production after 5 h). Synthesis of the M _r 23 000 and high M _r proteins appeared to be mutally exclusive, and to be regulated by the cell's iron status. Cells inoculated into medium containing 20 μM ferric citrate took up 92% of the iron within 1 h, suggesting the occurrence of a nonsiderophore mediated, 'low affinity' iron uptake pathway.     

MauE and MauD proteins are essential in methylamine metabolism of Paracoccus denitrificans

Synthesis of enzymes involved in methylamine oxidation via methylamine dehydrogenase (MADH) is encoded by genes present in the mau cluster. Here we describe the sequence of the mauE and mauD genes from Paracoccus denitrificans as well as some properties of mauE and mauD mutants of this organism. The amino acid sequences derived from the mauE and mauD genes showed high similarity with their counterparts in related methylotrophs. Secondary structure analyses of the amino acid sequences predicted that MauE is a membrane protein with five transmembrane-spanning helices and that MauD is a soluble protein with an N-terminal hydrophobic tail. Sequence comparison of MauD proteins from different organisms showed that these proteins have a conserved motif, Cys-Pro-Xaa-Cys, which is similar to a conserved motif found in periplasmic proteins that are involved in the biosynthesis of bacterial periplasmic enzymes containing haem c and/or disulphide bonds. The mauE and mauD mutant strains were unable to grow on methylamine but they grew well on other C₁-compounds. These mutants grown under MADH-inducing conditions contained normal levels of the natural electron acceptor amicyanin, but undetectable levels of the -subunit and low levels of the -subunit of MADH. It is proposed, therefore, that MauE and MauD are specifically involved in the processing, transport, and/or maturation of the -subunit and that the absence of each of these proteins leads to production of a non-functional -subunit which becomes rapidly degraded.     

Genetic tools for Paracoccus denitrificans

Abstract: Paracoccus denitrificans strain PD1222 (from N. Harms, Vrije Universiteit, Amsterdam, The Netherlands), which is deficient in a host-specific restriction system, could be successfully used for (1) transposon Tn5 mutagenesis, (2) phage P1-mediated generalized transduction and (3) construction of Hfr strains. Experimental protocols and some properties of an ammonium transport-deficient mutant are described.