Definition and distinction between assimilatory, dissimilatory and respiratory pathways.
Explore the source record for details and available documents.
Bacteria↗
Biomedical subjects
S J Ferguson
Publications and source records attributed to S J Ferguson.
Molecular genetics of the genus Paracoccus: metabolically versatile bacteria with bioenergetic flexibility.
Paracoccus denitrificans and its near relative Paracoccus versutus (formerly known as Thiobacilllus versutus) have been attracting increasing attention because the aerobic respiratory system of P. denitrificans has long been regarded as a model for that of the mitochondrion, with which there are many components (e.g., cytochrome aa3 oxidase) in common. Members of the genus exhibit a great range of metabolic flexibility, particularly with respect to processes involving respiration. Prominent examples of flexibility are the use in denitrification of nitrate, nitrite, nitrous oxide, and nitric oxide as alternative electron acceptors to oxygen and the ability to use C1 compounds (e.g., methanol and methylamine) as electron donors to the respiratory chains. The proteins required for these respiratory processes are not constitutive, and the underlying complex regulatory systems that regulate their expression are beginning to be unraveled. There has been uncertainty about whether transcription in a member of the alpha-3 Proteobacteria such as P. denitrificans involves a conventional sigma70-type RNA polymerase, especially since canonical -35 and -10 DNA binding sites have not been readily identified. In this review, we argue that many genes, in particular those encoding constitutive proteins, may be under the control of a sigma70 RNA polymerase very closely related to that of Rhodobacter capsulatus. While the main focus is on the structure and regulation of genes coding for products involved in respiratory processes in Paracoccus, the current state of knowledge of the components of such respiratory pathways, and their biogenesis, is also reviewed.
Base Sequence↗
Two enzymes with a common function but different heme ligands in the forms as isolated. Optical and magnetic properties of the heme groups in the oxidized forms of nitrite reductase, cytochrome cd1, from Pseudomonas stutzeri and Thiosphaera pantotropha.
It is shown that, in the oxidized state, heme c of Pseudomonas stutzeri (ZoBell strain) cytochrome cd1 has histidine-methionine ligation as observed for cytochrome cd1 from Pseudomonas aeruginosa [Sutherland, J., Greenwood, C., Peterson, J., and Thomson, A. J. (1986) Biochem. J. 233, 893-898]. However, the X-ray structure of Thiosphaera pantotropha cytochrome cd1 reveals bis-histidine ligation for heme c. It is confirmed by EPR and near-infrared (NIR) MCD measurements that the bis-histidine coordination remains unaltered in the solution phase. Hence, the difference between the heme c ligation states defines two distinct classes of oxidized cytochromes cd1 as isolated. A weak feature in the T. pantotropha NIR MCD at 1900 nm suggests that a small population of heme c has histidine-methionine coordination. The ligation state of heme d1 cannot be defined with the same level of confidence, because the porphyrin-to-Fe(III) charge-transfer (CT) bands are less well characterized for this class of partially reduced porphyrin ring. However, variable temperature absorption and MCD spectra show that, in the T. pantotropha enzyme, heme d1 exists in a thermal low-spin/high-spin mixture with the low-spin as the ground state, whereas in P. stutzeri cytochrome cd1, and d1 heme is low-spin at all temperatures. A weak band, assigned as the heme d1 porphyrin-pi(a1u,a2u)-to-ferric(d) charge-transfer transition has been identified for the first time at 2170 nm. Its magnetic properties show the heme d1 to have an unusual (dxz,yz)4(dxy)1 electronic ground state as is found for low-spin Fe(III) chlorins [Cheesman, M. R., and Walker, F. A. (1996) J. Am. Chem. Soc. 118, 7373-7380]. It is proposed that the localization of the Fe(III) unpaired d-electron in an orbital lying in the heme plane may decrease the affinity of the Fe(III) heme for unsaturated ligands such as NO. Although heme d1 in the enzymes from P. stutzeri and T. pantotropha shows different temperature-dependent spin properties, the positions of the low-spin Fe(III) alpha-absorption band, at approximately 640 nm, are very similar to those observed for cytochromes cd1 from eight other sources, suggesting that all have similar strength fields from the axial ligands and, hence, that all have the same coordination, namely histidine-tyrosine or possibly histidine-hydroxide at the heme.
Amino Acid Sequence↗
Pulse radiolysis studies on cytochrome cd1 nitrite reductase from Thiosphaera pantotropha: evidence for a fast intramolecular electron transfer from c-heme to d1-heme.
Electron transfer within cytochrome cd1 from Thiosphaera pantotropha was investigated by the technique of pulse radiolysis. The reduction of the heme centers in this nitrite reductase occurred in two phases as judged from kinetic difference spectra. In the faster phase, radiolytically generated N-methylnicotinamide (NMA) radicals selectively reduced the c-heme of the enzyme. From the absorbance increase at 420 nm, a characteristic of formation of the ferrousc-heme, the second-order rate constant for this electron transfer process was estimated to be 3.8 x 10(9) M-1 s-1 at pH 7.0. In the slower phase, a decrease of absorption around 420 and 550 nm, corresponding to a reoxidation of the c-heme, was accompanied by an increase of absorption around 460 and 640 nm, characteristic of formation of the reduced d1-heme. This indicated that an intramolecular electron transfer from the c-heme to the d1-heme occurred. The first-order rate constant of this process was calculated to be 1.4 x 10(3) s-1 at pH 7.0 and was independent of the enzyme concentration. In the presence of nitrite the interheme electron transfer rate was not affected, but on a time scale of seconds a new species associated with the d1-heme, having an absorption maximum at 640 nm, was detected and is proposed to reflect ligand binding to this heme. These results suggest the role of the c-heme as the electron acceptor site in cytochrome cd1 and in mediating the electron transfer to the catalytic site of the enzyme. Moreover, the fast interheme electron transfer rate argues against this process being the rate determining step in catalysis.
Cytochrome c Group↗
Haem-ligand switching during catalysis in crystals of a nitrogen-cycle enzyme.
Cytochrome cd1 nitrite reductase catalyses the conversion of nitrite to nitric oxide in the nitrogen cycle. The crystal structure of the oxidized enzyme shows that the d1 haem iron of the active site is ligated by His/Tyr side chains, and the c haem iron is ligated by a His/His ligand pair. Here we show that both haems undergo re-ligation during catalysis. Upon reduction, the tyrosine ligand of the d1 haem is released to allow substrate binding. Concomitantly, a refolding of the cytochrome c domain takes place, resulting in an unexpected change of the c haem iron coordination from His 17/His 69 to Met106/His69. This step is similar to the last steps in the folding of cytochrome c. The changes must affect the redox potential of the haems, and suggest a mechanism by which internal electron transfer is regulated. Structures of reaction intermediates show how nitric oxide is formed and expelled from the active-site iron, as well as how both haems return to their starting coordination. These results show how redox energy can be switched into conformational energy within a haem protein.
Catalysis↗
Cytochrome cd1 structure: unusual haem environments in a nitrite reductase and analysis of factors contributing to beta-propeller folds.
The central tunnel of the eight-bladed beta-propeller domain of cytochrome cd1 (nitrite reductase) is seen, from a 1.28 A resolution structure, to contain hydrogen donors and acceptors that are satisfied by interaction either with water or the d1 haem. The d1 haem, although bound by an extensive network of hydrogen bonds, is not distorted in its binding pocket and is confirmed to have exactly the dioxoisobacteriochlorin structure proposed from chemical studies. A biological rationale is advanced for the undistorted structure of the d1 haem and the large number of hydrogen bonds it makes. The beta-propeller domain can be closely superimposed on that of methanol dehydrogenase despite the enzymes sharing no common sequence motifs and using a different set of interactions to "Velcro" close the propeller. The sequence and likely structural relationships between cytochrome cd1 or methanol dehydrogenase and other predicted eight-bladed beta-propeller domains in proteins, such as the pyrolloquinoline quinone-dependent alcohol dehydrogenase, are discussed and compared with other propeller proteins. From sequencing the nirS gene of Thiosphaera pantotropha, it is established that the amino acid sequence deduced previously in part from X-ray diffraction data at lower resolution was largely correct, as was the proposal that eight N-terminal amino acid residues were not seen in the structure. The unusual haem iron environments in both the c-type cytochrome domain, with His/His coordination, and the d1-type cytochrome domain with Tyr/His coordination are related to the functions of the redox centres.
Amino Acid Sequence↗
The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli.
The pseudoazurin gene from Thiosphaera pantotropha has been cloned and sequenced. The deduced amino acid sequence showed that the protein contains an unusually alanine-rich signal peptide, 22 amino acid residues in length, which targets the protein to the periplasm. This pseudoazurin was expressed in large amounts in the periplasm of Escherichia coli when the gene with its native ribosome-binding site was placed downstream of the lac promoter. Removal of a putative hairpin-forming structure upstream of the ribosome-binding site increased the yield of the purified protein to approximately 80 mg/l. The recombinant protein is indistinguishable from that purified from its natural host. A primer extension study indicated that the pseudoazurin structural gene (pazS) is under the control of the Fnr/Nnr regulatory system, but no promoter-binding sequence could be recognized. The amino acid sequence of pseudoazurin from Paracoccus denitrificans is also reported.
Amino Acid Sequence↗
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 aa3, 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 aa3 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.
Alcohol Oxidoreductases↗
Unexpected implications from the Helicobacter pylori genome for understanding periplasmic c-type cytochrome assembly in gram-negative bacteria in coexistence with disulphide bond formation.
Explore the source record for details and available documents.
Cytochrome c Group↗
Cytokines in human breast milk.
In early life humans derive all their nutrients from milk. Human milk provides the neonate with not only a food source but also a source of biologically active molecules such as hormones, growth factors and cytokines. Cytokines are multifunctional glycoproteins involved in cell communication and immune system activation. Two of these cytokines, granulocyte-colony stimulating factor (G-CSF) and interleukin-6 (IL-6), are present in variable concentrations in human milk. We measured G-CSF and IL-6 concentrations in healthy nursing mothers. The 30 milk samples assayed contained G-CSF levels from 14 pg/mL to > 2500 pg/mL. Of the 28 milk samples assayed for IL-6, 24 contained levels from 0.81 pg/mL to 306 pg/ mL. The remainder had levels below the detection limit of the assay. These biologically active molecules may play a role in neonatal growth and development, and may also protect the infant against infection.
Granulocyte Colony-Stimulating Factor↗
Mutants of Escherichia coli lacking disulphide oxidoreductases DsbA and DsbB cannot synthesise an exogenous monohaem c-type cytochrome except in the presence of disulphide compounds.
Absence through mutation of two proteins involved in periplasmic disulphide bond formation, DsbA and DsbB, results in failure of anaerobically grown Escherichia coli to synthesise the holo forms of either its endogenous c-type cytochrome nitrite reductase or exogenous cytochrome c550 from Paracoccus denitrificans. The synthesis of both cytochromes can be restored to the mutants by inclusion in the growth media of compounds containing disulphide bonds, e.g., the oxidised form of glutathione. The results suggest that the attachment of haem to the CXXCH motif of a periplasmic c-type cytochrome may be preceeded by the formation of one or more intra- or intermolecular disulphide bonds involving the cysteine residues of this motif.
Alkaline Phosphatase↗
Structural investigation of the molybdenum site of the periplasmic nitrate reductase from Thiosphaera pantotropha by X-ray absorption spectroscopy.
The molybdenum centre of the periplasmic respiratory nitrate reductase from the denitrifying bacterium Thiosphaera pantotropha has been probed using molybdenum K-edge X-ray absorption spectroscopy. The optimum fit of the Mo(VI) EXAFS suggests two ==O, three -S- and either a fourth -S- or an -O-/-N- as molybdenum ligands in the ferricyanide-oxidized enzyme. Three of the -S- ligands are proposed to be the two sulphur atoms of the molybdopterin dithiolene group and Cys-181. Comparison of the EXAFS of the ferricyanide-oxidized enzyme with that of a nitrate-treated sample containing 30% Mo(V) suggests that the Mo(VI)-->Mo(V) reduction is accompanied by conversion of one ==O to -O-. The best fit to the Mo(IV) EXAFS of dithionite-reduced enzyme was obtained using one ==O, one -O- and four -S-/-Cl ligands. The periplasmic nitrate reductase molybdenum co-ordination environment in both the Mo(VI) and Mo(IV) oxidation states is distinct from that found in the membrane-bound respiratory nitrate reductase.
Amino Acid Sequence↗
Finite element stress analysis of a hybrid fracture fixation plate.
Metal plates are commonly used in the operative treatment of bone fractures. Rigid metal plates stabilize the fracture site, maintain good contact between bone fragments and allow early weight bearing and patient mobility. However, treatment with rigid metal plates can cause localized bone atrophy due to stress-shielding and interference with blood circulation, and the weakened bone can refracture after plate removal. A hybrid bone plate system that combines the torsional and bending rigidity of a metal plate with the axial compliance of a polymer insert has been designed. A three-dimensional, quarter-symmetric finite element model was generated for a canine femur diaphysis plated with this metal/polymer hybrid design. A model with a standard metal fixation plate was also generated for comparison purposes. The stress state in the underlying bone was examined for several loading conditions taken from published in vivo studies. The finite element model was used to study the performance of biodegradable polymer inserts in the plate system. The flexible plate reduced stress-shielding effects at the fracture site when subjected to an axial load. The bending strength of the plate was not compromised by the addition of the polymer inserts. Biodegradable inserts further enhanced the performance of the new plate design, transferring less of the axial load to the plate as the inserts broke down.
Animals↗
Cytochrome c550 expression in Paracoccus denitrificans strongly depends on growth condition: identification of promoter region for cycA by transcription start analysis.
The periplasmic cytochrome c550 content of Paracoccus denitrificans has been shown by immunological detection to be strongly dependent on the mode of growth. Cells grown under anaerobic, denitrifying conditions or methylotrophically in the presence of oxygen contained substantially more cytochrome c550 than cells grown aerobically on multicarbon substrates. A similar pattern was observed when expression of the cycA gene (encoding cytochrome c550), was monitored using an Escherichia coli alkaline phosphatase gene (phoA) fusion as a reporter of cycA promoter activity. The increase in cycA expression observed during growth on C1 substrates was substantially diminished if succinate was also present. These results reveal that expression of cycA is subject to multiple regulatory controls and suggest that cytochrome c550 has a general role in electron transfer to periplasmic reductases required for anaerobic denitrifying growth and from dehydrogenases required for aerobic growth on C1 compounds. Two major transcriptional initiation start points for the cycA gene have been identified.
Base Sequence↗
Alteration of haem-attachment and signal-cleavage sites for Paracoccus denitrificans cytochrome C550 probes pathway of c-type cytochrome biogenesis in Escherichia coli.
Paracoccus denitrificans cytochrome C550 is expressed as a periplasmic holo-protein in Escherichia coli; amino acid substitutions of cysteine residues in the haem-binding motif (Cys-X-X-Cys-His), either together or singly, prevented covalent attachment of haem but not polypeptide translocation into the periplasm. When the three alanine residues at positions -3 to -1 in the native signal-cleavage site were deleted, or alanine at -1 was changed to glutamine, signal cleavage was at alternative sites (after only ten residues in the latter case), but haem attachment still occurred. When the same three alanines were changed to Asp-Glu-Asp, a membrane-associated apo product that had retained the complete signal sequence was detected. These and other results presented here indicate that (i) haem attachment is not required for the apo-cytochrome C550 export to the periplasm; (ii) haem cannot attach to apo-cytochrome C550 when attached to the cytoplasmic membrane, suggesting that signal-sequence cleavage precedes periplasmic haem attachment, which can occur at as few as six residues from the mature N-terminus; and (iii) two cysteines are required for haem attachment, possibly because a disulphide bond is an intermediate. The gene for Saccharomyces cerevisiae mitochondrial iso-1-cytochrome c was expressed as a holo-protein in E. coli when fused with the signal sequence plus the first 10 residues of the mature cytochrome C550, indicating that the E. coli cellular apparatus for the c-type cytochrome biogenesis has a broad substrate specificity.
Amino Acid Sequence↗
Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions.
Explore the source record for details and available documents.
Amino Acid Sequence↗
The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha.
The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced. The large and small subunits of the enzyme are coded by napA and napB. The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor. Cysteine-181 is proposed to ligate the molybdenum atom. It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation. napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family. NapC may be the direct electron donor to the NapAB complex. napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.
Amino Acid Sequence↗
The complete amino acid sequence confirms the presence of pseudoazurin in Thiosphaera pantotropha.
The complete amino acid sequence, obtained by direct protein sequencing, of the pseudoazurin from Thiosphaera pantotropha is reported. It shows sequence identities varying from 46 to 66% with previously sequenced pseudoazurins. Previously identified conserved residues with key functions in pseudoazurins are found in the protein from T. pantotropha with the exception of glycine-39, the carbonyl group of which has been considered as a ligand to the copper, which is replaced by a serine residue. Electrospray-ionization MS (ESI-MS) has shown that pseudoazurin from T. pantotropha often contains two polypeptide species differing in molecular mass by 16 Da, presumably owing to oxidation of a methionine residue to a sulphoxide derivative. These two species have different endoproteinase Arg-C digestion patterns. Conditions for ESI-MS were identified that permitted either the retention or the loss of the single copper ion associated with the pseudoazurin. The aberrant tendency of T. pantotropha pseudoazurin to form a disulphide-bridged dimer on SDS/PAGE under some conditions is described.
Amino Acid Sequence↗