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S J Ferguson

Publications and source records attributed to S J Ferguson.

At least 91 records · Page 5Linked to original sources
PubMed1995

The anatomy of a bifunctional enzyme: structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cd1.

Cytochrome cd1-nitrite reductase is a bifunctional enzyme that catalyzes the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of oxygen to water. The 1.55 A crystal structure of the dimeric enzyme from Thiosphaera pantotropha is reported here. The protein was sequenced from the X-ray structure. Each subunit contains a covalent c heme with two axial His ligands (His-17, His-69) and a unique noncovalent d1 heme ligated by Tyr-25 and His-200. The d1 heme is the mononuclear iron center where both oxygen and nitrite reduction take place. The two types of heme are located in separate domains whose arrangement suggests a mechanism requiring domain movement during catalysis.

Amino Acid Sequence↗
PubMed1995

Pseudospecific docking surfaces on electron transfer proteins as illustrated by pseudoazurin, cytochrome c550 and cytochrome cd1 nitrite reductase.

The structure of pseudoazurin from Thiosphaera pantotropha has been determined and compared to structures of both soluble and membrane-bound periplasmic redox proteins. The results show a matching set of unipolar, but promiscuous, docking motifs based on a positive hydrophobic surface patch on the electron shuttle proteins pseudoazurin and cytochrome c550 and a negative hydrophobic patch on the surface of their known redox partners. The observed electrostatic handedness is argued to be associated with the charge-asymmetry of the membrane-bound components of the redox chain due to von Heijne's 'positives-inside' principle. We propose a 'positives-in-between' rule for electron shuttle proteins, and expect a negative hydrophobic patch to be present on both the highest and lowest redox potential species in a series of electron carriers.

Azurin↗
PubMed1995

Cloning and sequence analysis of cycH gene from Paracoccus denitrificans: the cycH gene product is required for assembly of all c-type cytochromes, including cytochrome c1.

A transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test. P. denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c-type cytochromes and accumulated periplasmic apocytochrome c550, but the membrane-bound holocytochromes c1 and c552 were present at 5-10% of the levels observed in wild-type cells. DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P. denitrificans wild-type DNA library. A hybridizing 3.05 kb BamHI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced. One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum. Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected. Significant differences between the phenotypes of P. denitrificans DP108 and the B. japonicum cycH mutant COX3, especially with respect to cytochrome c1 synthesis, suggest that the cycH gene product may be an assembly factor.

Amino Acid Sequence↗
PubMed1995

Sequence analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron-carrying arm of a redox loop.

Three genes, narH, narJ and narI, of the membrane-bound nitrate reductase operon of the denitrifying bacterium Thiosphaera pantotropha have been identified and sequenced. The derived gene products show high sequence similarity to the equivalent (beta, putative delta and gamma) subunits of the two membrane-bound nitrate reductases of the enteric bacterium Escherichia coli. All iron-sulphur cluster ligands proposed for the E. coli beta subunits are conserved in T. pantotropha NarH. Secondary structure analysis of NarJ suggests that this protein has a predominantly alpha-helical structure. Comparison of T. pantotropha NarI with the b-haem-binding integral membrane subunits of the E. coli enzymes allows assignment of His-53, His-63, His-186 and His-204 (T. pantotropha NarI numbering) as b-haem axial ligands and the construction of a three-dimensional model of this subunit. This model, in which the two b-haems are in different halves of the membrane bilayer, is consistent with a mechanism of energy conservation whereby electrons are moved from the periplasmic to the cytoplasmic side of the membrane via the haems. Similar movement of electrons is required in the membrane-bound uptake hydrogenases and membrane-bound formate dehydrogenases. We have identified two pairs of conserved histidine residues in the integral membrane subunits of these enzymes that are appropriately positioned to bind one haem towards each side of the membrane bilayer. One subunit of a hydrogenase complex involved in transfer of electrons across the cytoplasmic membrane of sulphate-reducing bacteria has structural resemblance to NarI.

Amino Acid Sequence↗
PubMed1995

Marriage timing of Chinese American and Japanese American women.

"Using U.S. census data, this study examines the differences in marriage rates and timing among White, Chinese American, and Japanese American women. An accelerated time model estimates the duration until marriage for each racial-ethnic group while controlling for nativity, education, birth cohort, ancestry, and English proficiency. Results show that White women have the shortest duration until marriage, with a smaller percentage remaining never married. Chinese American and Japanese American women delay first marriage longer and have higher percentages of never married women."

Americas↗
PubMed1994

Mo(V) electron paramagnetic resonance signals from the periplasmic nitrate reductase of Thiosphaera pantotropha.

A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphaera pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane-bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrificans. We have identified the Mo(V) species most likely to be the catalytically relevant one and characterised two other sets of Mo(V) EPR signals. As well as exhibiting EPR signals with g values typical of bacterial molybdenum-containing reductases, molybdenum-hydroxylase-like EPR signals can be elicited in the nitrate reductase of T. pantotropha upon treatment with excess dithionite. The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus. A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state. The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed.

Azotobacter↗
PubMed1994

Specific thiol compounds complement deficiency in c-type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane-bound disulphide isomerase-like protein.

Escherichia coli JCB606 carries a mutation in the dipZ gene, known to code for a disulphide isomerase-like protein, with the consequence that holo forms of neither exogenous nor endogenous c-type cytochromes are synthesised. This failure has been overcome by adding compounds containing thiol groups to the growth medium. Only L-cysteine and 2-mercaptoethane sulphonic acid were effective, suggesting a (stereo)specific binding site that could be occupied by these compounds in the absence of the catalytic domain of DipZ.

Alkaline Phosphatase↗
PubMed1994

Characterization of the paramagnetic iron-containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase.

Electron paramagnetic resonance spectroscopy signals attributable to low-spin haem c in the oxidised protein and [4Fe-4S]1+ in the dithionite-reduced protein were identified, at low temperature, in Thiosphaera pantotropha periplasmic nitrate reductase. Spin integration of these signals as well as elemental analysis suggest a stoichiometry of 1.3-1.6 c-haem and 1 [4Fe-4S] cluster per enzyme molecule. The Em (at pH 7.4) of the [4Fe-4S]2+,1+ couple, -160 mV, means that it is unlikely to be physiologically reducible. Peptide sequences from the 90 kDa subunit indicate that the enzyme is a member of the family of molybdopterin guanine dinucleotide-binding polypeptides, the majority of which possess a putative [4Fe-4S] cluster binding sequence and thus may also bind a (low potential) iron-sulphur cluster.

Amino Acid Sequence↗
PubMed1994

A mutation blocking the formation of membrane or periplasmic endogenous and exogenous c-type cytochromes in Escherichia coli permits the cytoplasmic formation of Hydrogenobacter thermophilus holo cytochrome c552.

A mutant of Escherichia coli, JCB606, shown to be pleiotropically deficient in the formation of endogenous membrane and periplasmic c-type cytochromes, synthesised the apo form of the exogenous cytochrome c550 from Paracoccus denitrificans, but not the holo form. In contrast, a cytoplasmically located holo form of Hydrogenobacter thermophilus cytochrome c552 was found in E. coli JCB606. These findings support the proposition that the formation of the cytoplasmic H. thermophilus cytochrome c552 in E. coli does not involve the physiological pathway of c-type cytochrome biosynthesis in E. coli and that the haem insertion may be catalysed.

Cytochrome c Group↗
PubMed1994

Synthesis of holo Paracoccus denitrificans cytochrome c550 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c552 does not. Implications for c-type cytochrome biogenesis.

Expression from a plasmid of the complete gene, including the codons for the N-terminal periplasmic targeting signal, for cytochrome c550 of Paracoccus denitrificans led to the formation of the holo protein in the periplasms of both P. denitrificans and Escherichia coli. Expression of the gene from which the region coding for the targeting signal had been specifically deleted resulted in formation of apo-protein in the cytoplasms of both organisms. These findings are consistent with haem attachment occurring in the periplasm. In contrast, the formation of holo cytochrome c552 from Hydrogenobacter thermophilus following expression of the gene lacking the periplasmic targeting sequence in either P. denitrificans or E. coli is attributed to spontaneous cytoplasmic attachment of haem to the thermostable protein.

Bacteria, Aerobic↗
PubMed1994

Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.

Cell Membrane↗
PubMed1994

Characterisation and amino acid sequence of cytochrome c-550 from Thiosphaera pantotropha.

A cytochrome c-550, with mid-point potential +265 mV, has been purified from Thiosphaera pantotropha. The cytochrome was recognised by antibodies to Paracoccus denitrificans cytochrome c-550, but the two proteins were not immunologically identical. Amino acid sequencing of the cytochrome c-550 showed 85.9% and 95.5% identities, respectively, with the cytochromes c-550 of P. denitrificans and Thiobacillus versutus; these are amongst the highest values reported for similarities between class I c-type cytochromes of the c2 group. These similarities are consistent with the published values of 85% for the overall DNA similarity of P. denitrificans and T. pantotropha, but contrast with published 16S rRNA analyses which indicate identity between T. pantotropha and P. denitrificans and 97.5% similarity of T. versutus with these two organisms. Analysis by plasma-desorption mass spectrometry of the peptide containing the haem-binding motif isolated from the apocytochrome has shown that an Hg atom binds to one or both of the two thiol groups.

Amino Acid Sequence↗
PubMed1994

Denitrification and its control.

Denitrification in bacteria comprises a series of four reduction reactions; for nitrate, nitrite, nitric oxide and nitrous oxide. Nitrogen gas is the final product. The nature of the enzymes catalysing these reactions is described along with the the properties of the underlying electron transport systems. The factors influencing the expression of the reductases for the four reactions are reviewed along with the effect of oxygen on the activities of the enzymes of denitrification. The main emphasis is on observations made with Paracoccus denitrificans and Pseudomonas stutzeri.

Electron Transport↗
PubMed1994

Differential reduction in soluble and membrane-bound c-type cytochrome contents in a Paracoccus denitrificans mutant partially deficient in 5-aminolevulinate synthase activity.

A mutant of Paracoccus denitrificans, DP104, unable to grow anaerobically with nitrate as the terminal electron acceptor or aerobically with methanol as the electron donor and staining negatively in the dimethylphenylene diamine oxidation (Nadi) test, was isolated by transposon Tn5::phoA mutagenesis. P. denitrificans DP104 grown aerobically with succinate or choline had very low levels (2 to 3% of the wild-type levels) of spectroscopically detectable soluble c-type cytochromes. In contrast, membrane cytochromes of the a, b, and c types were present at 50% of the levels found in the wild type. The apo form of cytochrome c550, at an approximately 1:1 molar ratio with the holo form, was found in the periplasm of DP104. The TnphoA element was shown to be inserted immediately upstream of the translational start of hemA, the gene coding for 5-aminolevulinate synthase, which was sequenced. Low-level expression of this gene, driven off an incidental promoter provided by TnphoA-cointegrated suicide vector DNA, is the basis of the phenotype which could be complemented by the addition of 5-aminolevulinate to growth media. Disruption of the hemA gene generated a P. denitrificans strain auxotrophic for 5-aminolevulinate, establishing that there is no hemA-independent pathway of heme synthesis in this organism. The differential deficiency in periplasmic c-type cytochromes relative to membrane cytochromes in DP104 is suggested to arise from unequal competition for the restricted supply of heme which results from the effects of the transposon insertion.

5-Aminolevulinate Synthetase↗
PubMed1993

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c-type cytochrome biogenesis synthesise the methylamine-dehydrogenase polypeptides but cannot assemble the tryptophan-tryptophylquinone group.

Five mutants of Methylobacterium extorquens and four mutants of Paracoccus denitrificans that have a general defect in c-type cytochrome synthesis also failed to assemble an active methylamine dehydrogenase. In all cases methanol dehydrogenase, another periplasmic enzyme, was fully active. All nine mutant strains accumulated both the heavy and light subunits of methylamine dehydrogenase to essentially wild-type levels. In all nine mutants, the heavy-subunit and light-subunit polypeptides were proteolytically processed, suggesting that translocation to the periplasm had occurred; in the case of the P. denitrificans mutants, a periplasmic location for the heavy and light subunits was confirmed experimentally. While specific quinone staining of the methylamine dehydrogenase light subunit in wild-type M. extorquens and P. denitrificans strains could readily be demonstrated, the light subunit polypeptides accumulated by the mutants did not quinone stain, indicating that the methylamine dehydrogenase prosthetic group, tryptophan tryptophylquinone, is not assembled in the absence of functional c-type cytochromes.

Benzoquinones↗