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PubMed2005

Miniaturized kinetic growth inhibition assay with denitrifying bacteria Paracoccus denitrificans.

A method to cultivate anaerobic bacteria on standard 96-well microplates with automatic recording of growth curves is presented. The method was used as a kinetic growth inhibition assay with denitrifying bacteria Paracoccus denitrificans, and applied to various heavy metal ions and selected agrochemicals. Incorporated in a battery of other biotest the assay could take into account effects of toxicants on denitrifying organisms. The results (EC50) revealed that the assay was relatively sensitive. Performed in vials, the assay was also applied to toxicity testing of volatile compounds and represented a convenient method for assessing samples containing volatile constituents.

Agrochemicals↗

PubMed2004

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase.

The oxidized "as isolated" form of Paracoccus pantotrophus cytochrome cd1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d1 heme. This form of the enzyme has previously been shown to be kinetically incompetent. Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated. Here, we show that P. pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, "as isolated" cytochrome cd1. NapC is the first protein from P. pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner.

Catalysis↗

PubMed2005

Thermal stability of outer membrane protein porin from Paracoccus denitrificans: FT-IR as a spectroscopic tool to study lipid-protein interaction.

Lipid protein interactions play a key role in the stability and function of various membrane proteins. Earlier we have reported the extreme thermal stability of porin from Paracoccus denitrificans reconstituted into liposomes. Here, we used Fourier transform infrared spectroscopy for a label free analysis of the global secondary structural changes and local changes in the tyrosine microenvironment. Our results show that a mixed lipid system (non-uniform bilayer) optimizes the thermal stability of porin as compared to the porin in pure lipids (uniform bilayer) or detergent micelles. This is in line with the fact that the bacterial outer membrane is a dynamic system made up of lipids of varying chain lengths, head groups and the barrel wall height contacting the membrane is uneven.

Cloning, Molecular↗

PubMed2006

Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples.

The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.

Cytochrome c Group↗

PubMed2006

Cloning and characterization of the astaxanthin biosynthesis gene cluster from the marine bacterium Paracoccus haeundaensis.

Carotenoids are important natural pigments produced by many microorganisms and plants. In a previous study, we isolated and characterized a marine bacterium, Paracoccus haeundaensis, which produces carotenoids, mainly astaxanthin. In the present study, a carotenoid biosynthesis gene cluster involved in the production of astaxanthin was isolated and characterized from this marine bacterium. The cluster contained six carotenogenic genes, five of which, with the same orientation, were designated crtW, crtZ, crtY, crtI, and crtB, and one of which, with the opposite orientation, was designated crtE. The crtW, crtZ, crtY, crtI, crtB, and crtE genes contained 726, 486, 1158, 1503, 912, and 879 base pairs, respectively, and encoded polypeptides of 242, 162, 386, 501, 304, and 293 amino acid residues, respectively. The stop codon of each crt gene, except crtE, overlapped the start codon of the following crt gene. Comparisons of the structures and nucleotide sequences of the crt genes showed that the signature domains of the carotenoid biosynthesis genes are highly conserved. The molecular phylogenetic tree analysis showed the evolutionary relationships among the carotenoid biosynthesis genes of various organisms. An expression vector (pCR-XR-TOPO-Crt-full vector) containing the astaxanthin biosynthesis genes was constructed and transformed into Escherichia coli BL21(DE3) Codon Plus cells. The E. coli transformants produced 400 microg astaxanthin per gram of dry cell weight.

Base Sequence↗

PubMed2005

Structure of the cytochrome complex SoxXA of Paracoccus pantotrophus, a heme enzyme initiating chemotrophic sulfur oxidation.

The sulfur-oxidizing enzyme system (Sox) of the chemotroph Paracoccus pantotrophus is composed of several proteins, which together oxidize hydrogen sulfide, sulfur, thiosulfate or sulfite and transfers the gained electrons to the respiratory chain. The hetero-dimeric cytochrome c complex SoxXA functions as heme enzyme and links covalently the sulfur substrate to the thiol of the cysteine-138 residue of the SoxY protein of the SoxYZ complex. Here, we report the crystal structure of the c-type cytochrome complex SoxXA. The structure could be solved by molecular replacement and refined to a resolution of 1.9A identifying the axial heme-iron coordination involving an unusual Cys-251 thiolate of heme2. Distance measurements between the three heme groups provide deeper insight into the electron transport inside SoxXA and merge in a better understanding of the initial step of the aerobic sulfur oxidation process in chemotrophic bacteria.

Amino Acid Sequence↗

PubMed2005

Cloning and characterization of a region responsible for the maintenance of megaplasmid pTAV3 of Paracoccus versutus UW1.

Using cointegrate formation, we constructed a basic replicon of the megaplasmid/mini-chromosome pTAV3 of Paracoccus versutus UW1. It is composed of two adjacent modules, responsible for plasmid replication (rep) and partitioning (par). Functional analysis of the par region identified a determinant of incompatibility (inc2), whose presence is crucial for proper partitioning (the partitioning site). Database searches revealed that the only known replicon with significant homology to that of pTAV3 is encoded by the chromosome cII of Rhodobacter sphaeroides 2.4.1. Incompatibility studies showed that closely related basic replicons are also encoded by megaplasmids (above 400 kb) harbored by four strains of P. pantotrophus. Basic replicons of the pTAV3-type are able to maintain large bacterial genomes, therefore they appear to be good candidates for the construction of vectors specific for Alphaproteobacteria.

Base Sequence↗

PubMed2006

Activation and catalysis of the di-heme cytochrome c peroxidase from Paracoccus pantotrophus.

Bacterial cytochrome c peroxidases contain an electron transferring (E) heme domain and a peroxidatic (P) heme domain. All but one of these enzymes are isolated in an inactive oxidized state and require reduction of the E heme by a small redox donor protein in order to activate the P heme. Here we present the structures of the inactive oxidized and active mixed valence enzyme from Paracoccus pantotrophus. Chain flexibility in the former, as expressed by the crystallographic temperature factors, is strikingly distributed in certain loop regions, and these coincide with the regions of conformational change that occur in forming the active mixed valence enzyme. On the basis of these changes, we postulate a series of events that occur to link the trigger of the electron entering the E heme from either pseudoazurin or cytochrome c(550) and the dissociation of a coordinating histidine at the P heme, which allows substrate access.

Amino Acid Sequence↗

PubMed2003

Inhibition by phenylglyoxal of nitrate transport in Paracoccus denitrificans: a comparison with the effect of a protonophorous uncoupler.

The amino acid modifier phenylglyoxal (PG) gradually inactivated the methyl viologen-coupled nitrate reductase activity of the anoxically grown whole cells of Paracoccus denitrificans. A double log plot of the pseudo-first-order inactivation rate constant versus PG concentration was linear with a mean slope of 1.4 (0.1M sodium phosphate) or 0.87 (0.1M sodium borate). Phenylglyoxalation of cells lowered the limiting velocity (V), while hardly affecting the apparent half-saturation concentration (K(m)) of nitrate. Nitrate afforded no protection against inactivation. The inhibition by PG could be removed by the detergent Triton X-100 or by the lipid-soluble tetraphenylphosphonium countercation, suggesting that PG exerts its effect at the level of nitrate transport. Based on studies with membrane potential- and pH-sensitive fluorescent probes, the inhibition was shown not to be due to changes in the electrochemical gradient of hydrogen ions. Both K(m) and V values for nitrate uptake increased in a hyperbolic fashion in response to exogenously added nitrite. Nitrite promoted a bypass of the inhibition caused by low concentrations of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP), but was almost ineffective in the case of the PG block. These results are rationalized in terms of two nitrate import pathways that are comparably inhibited by PG and differ in their sensitivities to CCCP. A simplified kinetic model for phenylglyoxalation is proposed to account for the observed nonintegral reaction orders.

Anaerobiosis↗

PubMed2002

Succinate:quinone oxidoreductase in the bacteria Paracoccus denitrificans and Bacillus subtilis.

An overview of the present knowledge about succinate:quinone oxidoreductase in Paracoccus denitrificans and Bacillus subtilis is presented. P. denitrificans contains a monoheme succinate:ubiquinone oxidoreductase that is similar to that of mammalian mitochondria with respect to composition and sensitivity to carboxin. Results obtained with carboxin-resistant P. denitrificans mutants provide information about quinone-binding sites on the enzyme and the molecular basis for the resistance. B. subtilis contains a diheme succinate:menaquinone oxidoreductase whose activity is dependent on the electrochemical gradient across the cytoplasmic membrane. Data from studies of mutant variants of the B. subtilis enzyme combined with available crystal structures of a similar enzyme, Wolinella succinogenes fumarate reductase, substantiate a proposed explanation for the mechanism of coupling between quinone reductase activity and transmembrane potential.

Amino Acid Sequence↗

PubMed1991

Evidence for the role of soluble cytochrome c in the dissimilatory reduction of nitrite and nitrous oxide by cells of Paracoccus denitrificans.

The role of periplasmic cytochrome c in the denitrification pathway has been investigated using a wild-type and/or a cytochrome c deficient strain of Paracoccus denitrificans. The reconstitution experiments with the isolated proteins showed that bacterial cytochrome c-550 restored the electron transport from the cytoplasmic membrane to soluble nitrite reductase (cytochrome cd1). In response to decreased aeration lasting 3 h, the HUUG25 strain synthesized nitrous-oxide reductase significantly starved of electrons from the respiratory chain and only very small amounts of soluble cytochrome c. The membrane-bound part of the respiratory chain catalyzing the reduction of soluble cytochrome c resembled an autologous region in wild-type cells kinetically and by its sensitivity to antimycin. In the periplasmic fraction obtained from anaerobically grown wild-type cells N2O caused the reoxidation of endogenous cytochrome(s) c previously reduced by N,N,N',N' tetramethyl-p-phenylenediamine plus ascorbate. All these results indicate the involvement of soluble cytochrome(s) c as the electron donor(s) for the reduction of NO2- and N2O in the periplasmic space of cells.

Cytochrome c Group↗

PubMed1992

Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al. (1981) Biochim. Biophys. Acta 636, 162-167). In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein. A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA. The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme. The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane. One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well. Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O.

Copper↗

PubMed1989

Electron transport reactions in a cytochrome c-deficient mutant of Paracoccus denitrificans.

A mutant of Paracoccus denitrificans which is deficient in c-type cytochromes grows aerobically with generation times similar to those obtained with a wild-type strain. The aa3-type oxidase is functional in the mutant as judged by spectrophotometric assays of cytochrome c oxidation using the membrane particles and cytochrome aa3 reduction in whole cells. The cytochrome c oxidase (aa3-type) of the c-less mutant oxidizes soluble cytochrome c at rates equivalent to those obtained with the wild-type. NADH and succinate oxidase activities of the membrane preparations of the mutant and wild-type are also comparable in the absence of detergent treatment. Exogenous soluble cytochrome c can be both reduced by NADH- and succinate-linked systems and oxidized by cytochrome aa3 present in membranes of the mutant strain. Rapid overall electron transport can occur in the c-less mutant, suggesting that reactions result from collision of diffusing complexes.

Cell Membrane↗

PubMed1996

Biochemical and spectroscopic properties of the four-subunit quinol oxidase (cytochrome ba3) from Paracoccus denitrificans.

The ba3 quinol oxidase from Paracoccus denitrificans has been purified by a new protocol leading to significantly higher yields than previously reported (Richter et al. (1994) J. Biol. Chem. 269, 23079-23086). In an SDS PAG an additional protein band compared with the previous preparation appears, which can be identified as the major form of subunit II. All protein bands can be assigned to genes of the qox operon by N-terminal sequencing, indicating that the oxidase consists of four subunits. In addition to one heme A, one heme B, and one copper atom, the preparation contains two ubiquinone molecules per enzyme. The oxidase is further characterized by electron paramagnetic resonance (EPR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy.

Circular Dichroism↗

PubMed1997

Purification and initial kinetic and spectroscopic characterization of NO reductase from Paracoccus denitrificans.

A new and relatively simple procedure to purify NO reductase from Paracoccus denitrificans by using the detergent lauryl maltoside has been developed. The purified enzyme consists of two subunits according to SDS polyacrylamide gel electrophoresis. Analysis of the content of prosthetic groups indicates the presence of non-haem iron in addition to the presence b and c cytochromes yielding a stoichiometry of haem b/haem c/non-haem iron = 2:1:1. The optical spectrum of reduced NO reductase shows bands of low-spin haem c and haem b with alpha-band absorbance maxima at 551 nm and 558 nm, respectively. The optical spectrum of oxidized NO reductase shows a broad absorbance hand around 590 nm which disappears upon reduction. This latter absorbance is ascribed to a high-spin haem b (charge-transfer) transition. The presence of high-spin haem b is also indicated by the shifts observed in the optical spectrum of oxidized NO reductase in the presence of NO or in the spectrum of reduced enzyme after addition of CO. The main features of the EPR spectrum of the oxidized enzyme are resonances from a highly anisotropic low-spin haem b (gz = 3.53) and from an anisotropic low-spin haem c with gz, y, x = 2.99, 2.28, 1.46, the two haems being present in an approximate 1:1 stoichiometry. Minor signals representing about 1% of the enzyme concentration due to high-spin haem b (g = 5.8-6.2) and a novel type of signal with g = 2.009 ascribed to high-spin non-haem ferric iron were also observed. The analysis of steady-state kinetic measurements of the NO reductase activity shows a sigmoidal relation between rate of NO reduction and NO concentration, consistent with a model describing sequential binding of two molecules of NO to the reduced enzyme. At high NO concentrations substrate inhibition occurs (Ki(apparent) = 13.5 microM) suggested to be due to binding of NO to oxidized enzyme. The absence from the EPR spectrum of signals originating from ferric non-haem iron and ferric high-spin haem b in stoichiometric amounts with respect to the enzyme concentration is suggested to be due to an antiferromagnetic coupling between these two centers. The steady-state kinetic behaviour and the optical and EPR spectroscopic properties of the NO reductase are incorporated into a tentative structural and mechanistic model.

Binding Sites↗

PubMed1998

Comparison of energization of complex I in membrane particles from Paracoccus denitrificans and bovine heart mitochondria.

The results of preliminary studies of the effects of energization on the catalytic and EPR properties of complex I in tightly coupled membrane vesicles of Paracoccus denitrificans (SPP) are presented. They are compared to those observed in submitochondrial particles from bovine heart (SMP). All signs of energization of complex I detected by EPR in SMP (uncoupler-sensitive splitting of the gz lines of the clusters 2 and a broadening of their gxy lines, a fast-relaxing, piericidin-sensitive ubiquinone-radical signal, and a broad signal around g = 1.94) were also observed with the bacterial enzyme. There were some prominent differences, though. The signal of the fast-relaxing radicals could be evoked both in the presence or absence of reduced clusters 2, suggesting that enhancement of its spin-relaxation rate is caused by coupling to another paramagnet. The signal was hardly affected by the presence of gramicidin. The slow-relaxing radical signal did not disappear upon anaerobiosis, but was detectable for at least another 30 s. The fast-relaxing signal vanished immediately upon anaerobiosis. The activity of the bacterial enzyme during oxidation of NADH by oxygen or reduction of NAD induced by succinate oxidation, was 5-6 times higher than that of the mitochondrial enzyme. Unlike the mitochondrial enzyme, the bacterial enzyme was not inactivated by incubation at 35 degrees C. The spin concentration of the NADH-reducible [2Fe-2S] cluster (1b) was half that of the clusters 2, indicating no difference with the mitochondrial enzyme.

Animals↗

PubMed1999

Evaluation of relative contributions of two enzymes supposed to metabolise hydrogen peroxide in Paracoccus denitrificans.

A biosensor exploiting an electrochemically mediated enzyme-catalysed reaction was used to quantify relative contributions of cytoplasmic catalase and periplasmic cytochrome c peroxidase to the overall rate of hydrogen peroxide breakdown in cells of Paracoccus denitrificans. The effects of antimycin (an inhibitor of electron flow to cytochrome c peroxidase), the reaction rate versus substrate concentration profiles for the whole cells and subcellular fractions, and the time courses of oxygen concentration demonstrated a profound decrease in the capacity of cytochrome c peroxidase to reduce H2O2 under in vivo conditions. The reason is suggested to be a competition for available electrons between the enzyme and terminal oxidases metabolising oxygen produced by catalase.

Biosensing Techniques↗

PubMed1999

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase.

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

Cloning, Molecular↗