PubMed Health⌕ Search

Biomedical subjects

R Cammack

Publications and source records attributed to R Cammack.

At least 73 records · Page 4Linked to original sources
PubMed1990

Interactions of iron-thiol-nitrosyl compounds with the phosphoroclastic system of Clostridium sporogenes.

Certain reagents, such as ascorbate or iron salts and thiols, enhance the bacteriostatic action of nitrite on food-spoilage bacteria. This may be due to the formation of nitric oxide and iron-thiol-nitrosyl [( Fe-S-NO]) complexes. The minimum concentrations of these reagents required to inhibit growth of Clostridium sporogenes were investigated. A mixture of nitrite (0.72 mM) with iron (1.44 mM) and cysteine (2.16 mM) was found to be extremely inhibitory when autoclaved and diluted into the culture medium. This mixture caused rapid cessation of growth and loss of cell viability at a final concentration corresponding to 40 microM-nitrite. If added to the initial culture medium, it prevented growth at 5 microM-nitrite. The mixture was more inhibitory, on the basis of the nitrite concentration used, than the 'Perigo factor', obtained by autoclaving nitrite in growth medium. [Fe-S-NO] compounds of known chemical structure were tested to determine if they were responsible for this effect. Total inhibition of cell growth was observed with the tetranuclear clusters [Fe4S3(NO)7] (Roussin's black salt), [Fe4S4(NO)4] or [Fe4Se3(NO)7], added at concentrations equivalent to 10 microM-nitrite, or with [Fe2(SMe)2(NO)4] (methyl ester of Roussin's red salt), equivalent to 200 microM-nitrite. The rate of hydrogen production in growing cell cultures was inhibited by [Fe4S3(NO)7] at levels equivalent to 2.5 microM-nitrite. EPR spectra of the inhibited cells showed features with g-values of 2.03, characteristic of mononuclear iron-nitrosyl species, and, under non-reducing conditions, an unusual signal at g = 1.65. There was no correlation between growth inhibition and the g = 2.03 signal, though there was a better correlation between inhibition and the g = 1.65 signal. The direct effects of the compounds were tested on the iron-sulphur proteins of the phosphoroclastic system, namely ferredoxin, pyruvate-ferredoxin oxidoreductase and hydrogenase. EPR spectra and enzyme assays showed that these proteins were not destroyed by [Fe4S3(NO)7], [Fe4S4(NO)4], [Fe2(SMe)2(NO)4], [Fe(SPh)2(NO)2], or M2 (an autoclaved mixture of 66 mM-cysteine, 3.6 mM-FeSO4 and 0.72 mM-NaNO2) at concentrations higher than those that caused total inhibition of cell growth. Inhibition of cells by [Fe-S-NO] compounds is unlikely to be due to interaction with the preformed enzymes. The possible formation of iron-nitrosyl complexes in vivo, and their inhibitory actions, are discussed.

Cell Division↗
PubMed1989

Electrochemical and spectroscopic characterization of the 7Fe form of ferredoxin III from Desulfovibrio africanus.

Desulfovibrio africanus ferredoxin III is a monomeric protein (Mr 6585) containing seven cysteine residues and 7-8 iron atoms and 6-8 atoms of acid-labile sulphur. It is shown that reversible unmediated electrochemistry of the two iron-sulphur clusters can be obtained by using a pyrolytic-graphite-'edge' carbon electrode in the presence of an appropriate aminoglycoside, neomycin or tobramycin, as promoter. Cyclic voltammetry reveals two well-defined reversible waves with E0' = -140 +/- 10 mV and -410 +/- 5 mV (standard hydrogen electrode) at 2 degrees C. Bulk reduction confirms that each of these corresponds to a one-electron process. Low-temperature e.p.r. and magnetic-c.d. spectroscopy identify the higher-potential redox couple with a cluster of core [3Fe-4S]1+.0 and the lower with a [4Fe-4S]2+.1+ centre. The low-temperature magnetic-c.d. spectra and magnetization properties of the three-iron cluster show that it is essentially identical with that in Desulfovibrio gigas ferredoxin II. We assign cysteine-11, -17 and -51 as ligands of the [3Fe-4S] core and cysteine-21, -41, -44 and -47 to the [4Fe-4S] centre.

Amino Acid Sequence↗
PubMed1989

Inhibition of Desulfovibrio gigas hydrogenase with copper salts and other metal ions.

The effect of several transition metals on the activity of Desulfovibrio gigas hydrogenase has been studied. Co(II) and Ni(II) at a concentration of 1 mM did not modify the activity of the enzyme; nor did they affect the pattern of activation/deactivation. Cu(II) inhibited the active hydrogenase, prepared by treatment with hydrogen, but had little effect on the 'unready' enzyme unless a reductant such as ascorbate was present, in which case inactivation took place either in air or under argon. Hg(II) also inactivated the enzyme irreversible in the 'unready' state without the requirement for reductants. The reaction of H2 uptake with methyl viologen was much more sensitive to inhibition than the H2/tritium exchange activity. EPR spectra of this preparation showed that the rates of decline were [3Fe-4S] signal greater than H2-uptake activity greater than Ni-A signal. Similar results were obtained when the protein was treated with Hg(II). The results demonstrate that the [3Fe-4S] cluster is not essential for H2-uptake activity with methyl viologen, but the integrity of [4Fe-4S] clusters is probably necessary to catalyze the reduction of methyl viologen with hydrogen. D. gigas hydrogenase was found to be highly resistant to digestion by proteases.

Copper↗
PubMed1989

Spectroscopic characterization of the nickel and iron-sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina. 1. Electron spin resonance spectroscopy.

The thermostable hydrogenase from Thiocapsa roseopersicina was examined by low-temperature ESR spectroscopy. Two types of signals were detected, from an oxidized iron-sulphur cluster and a nickel centre (Ni-A). In the oxidized protein additional signals were observed due to spin-spin interaction between the two paramagnetic centres. This interaction could be reversibly abolished by reduction to a redox potential below 105 mV. This implies that an additional redox centre is involved in the interaction, for which an Fe3+ ion is suggested. Reduction with hydrogen induced a second type of nickel ESR signal (Ni-C), corresponding to an intermediate redox state seen in other nickel hydrogenases. The Ni-C species was light-sensitive at cryogenic temperatures. At temperatures near to 4.2 K the Ni-C signal showed evidence of interaction with another paramagnetic centre, presumably a second iron-sulphur cluster. On reoxidation a signal due to a third Ni(III) species, Ni-B, increased in amplitude. These results establish that metal centres in the hydrogenase from T. roseopersicina are closely similar to those of the well-studied hydrogenase from Chromatium vinosum.

Chromatiaceae↗
PubMed1989

Spectroscopic characterization of the nickel and iron-sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina. 2. Electron spin-echo spectroscopy.

Pulsed electron-spin-resonance techniques were applied to the hydrogenase of the purple photosynthetic bacterium Thiocapsa roseopersicina, an enzyme which contains nickel and iron-sulphur clusters but no flavin. The linear electric field effect profile of the spectrum in the region of g = 2.01 indicated that the strong ESR signal in the oxidized protein is due to a [3Fe-4S] cluster. The electron spin-echo envelope of this spectrum was modulated by hyperfine interactions with 1H and 14N nuclei, probably from the polypeptide chain. The ESR spectrum of this species shows a complex pattern arising from spin-spin interaction with another paramagnet. When the protein was partially reduced by ascorbate plus phenazine methosulphate, the complexity of the spectrum was abolished but the form of the electron spin-echo envelope modulation (ESEEM) pattern was unchanged. This indicates that the reversible disappearance of the spin-spin interaction pattern on partial reduction is not due to cluster interconversion to a [4Fe-4S] cluster. In the ESR spectrum of nickel(III), weak hyperfine interactions with 1H and 14N were also observed by ESEEM. The nature of the interacting nuclei is discussed.

Chromatiaceae↗
PubMed1989

Isolation, characterization, and biological activity of the Methanococcus thermolithotrophicus ferredoxin.

A ferredoxin has been isolated from the thermophilic methanogen Methanococcus thermolithotrophicus. The native protein was a monomer exhibiting a molecular weight of 7,262, calculated from the amino acid composition. Its absorption spectrum had two maxima at 390 and 283 nm, with an absorbance ratio A390/A283 of 0.79. The absorption at 390 nm (E = 29 mM-1 cm-1) and the content of iron of the protein are in agreement with the presence of two 4Fe-4S clusters in M. thermolithotrophicus ferredoxin. Its amino acid composition showed the presence of eight cysteine residues, which is the required number of cysteines for the binding of two 4Fe-4S clusters. The protein was characterized by the lack of histidine, arginine, and leucine and a high content of valine. It was unusually stable to high temperatures but not to oxygen. The ESR spectrum of the protein in the oxidized state showed a minor signal at g = 2.01, corresponding to an oxidized 3Fe-4S cluster. The protein, which was difficult to reduce with dithionite or reduced mediators, exhibited in its reduced state a spectrum typical of two interacting reduced 4Fe-4S clusters. M. thermolithotrophicus ferredoxin functioned as an electron acceptor for the CO dehydrogenase complex with an extract free of ferredoxin. No reaction was detected with F420 or hydrogenase.

Amino Acids↗
PubMed1988

A pulsed EPR study of redox-dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase.

The nickel centre of hydrogenase from Desulfovibrio gigas was studied by electron spin echo envelope modulation (ESEEM) spectroscopy in the oxidized, unready (Ni-A) and H2-reduced active (Ni-C) states, both in H2O and 2H2O solutions. Fourier transforms of the 3-pulse ESEEM, taken at 8.7 GHz, for Ni-A and Ni-C in H2O contained similar peaks with narrow linewidths at frequencies of 0.4, 1.2 and 1.6 MHz, and a broader peak centered at 4.5 MHz. At 11.6 GHz, the low frequency components showed small field-dependent shifts, while the high frequency component was shifted to 5.1 MHz. These results are consistent with the presence of 14N, possibly from imidazole, coupled to the nickel centre. In 2H2O, Ni-A was shown to be inaccessible for exchange with solvent deuterons. In contrast, Ni-C was accessible to solvent exchange, with a deuterium population being in close proximity to the metal ion. Thus, the nickel environment of the active protein is different from that in the oxidized or unready state. On illumination of Ni-C, although EPR changes are seen, 14N coupling remains, and for the 2H2O sample, deuterium coupling is also retained.

Desulfovibrio↗
PubMed1988

Electron spin-echo spectroscopic studies of Escherichia coli fumarate reductase.

Electron spin-echo envelope modulation (ESEEM) spectroscopy was applied to the study of reduced Centre 1 of Escherichia coli fumarate reductase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1). The ESEEM spectrum derived from stimulated (3-pulse) echo envelopes obtained at 8.8 GHz contained lines at 0.9, 2.1, 3.0 and 4.2 MHz in the g = 1.94 region. When studied at 11.4 GHz, these low-frequency components scale with magnetic field in a manner indicating interaction between the unpaired electron spin of the Fe-S cluster and a weakly coupled 14N nucleus. Spectral simulations of these ESEEM data yield nuclear quadrupole interaction parameters indicative of peptide nitrogen. For oxidized protein, the magnetic-field dependence of the linear electric-field effect (LEFE) for Centre 3 was measured, and the results confirm the presence of a [3Fe-4S] cluster in the protein.

Electron Spin Resonance Spectroscopy↗
PubMed1988

Primary structure of protein B from Pseudomonas putida, member of a new class of 2Fe-2S ferredoxins.

The primary structure of the 2Fe-2S ferredoxin (protein B) from the benzene dioxygenase system of Pseudomonas putida strain NCIB 12190 was determined by gas-phase sequencing of the protein and its fragments. Fast atom bombardment mass spectrometry indicated a molecular mass of 11,860 Da. The sequence contained five cysteine residues, four of which would be required to coordinate the iron-sulphur cluster. The amino acid sequence determined in the present study is compared to that of a protein deduced from the DNA sequence from another strain of Pseudomonas putida. Little sequence homology was observed when protein B was compared to 2Fe-2S ferredoxins from plant and cyanobacterial sources. The novel sequence determined here suggests a new class of ferredoxin, which is consistent with the observed mid-point redox potential being significantly less negative (-155 mV) than those of the 2Fe-2S ferredoxins involved in photosynthesis (-310 to -455 mV).

Amino Acid Sequence↗
PubMed1987

Comparison of the spin-lattice relaxation properties of the two classes of [2Fe-2S] clusters in proteins.

Two classes of [2Fe-2S] proteins have been defined according to the mean value gav of their g tensor components (Bertrand, P., Guigliarelli, B., Gayda, J.P., Beardwood, P. and Gibson, J.F. (1985) Biochim. Biophys. Acta 831, 261-266). To characterize their magnetic properties better, we have compared the spin-lattice relaxation behavior of typical proteins which belong to these two classes, namely Spirulina maxima and adrenal ferredoxin for the gav approximately 1.96 class, Thermus thermophilus Rieske protein and Pseudomonas putida benzene dioxygenase for the gav approximately 1.91 class. For all these proteins, the data support the existence of an efficient Orbach process in the highest temperature range, which allows the determination of the exchange coupling parameter, J. From the comparison of the J values obtained in each class, it is concluded that the structural factors which determine the value of the g tensor and the strength of the antiferromagnetic exchange interactions are different.

Animals↗
PubMed1986

Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe-4S]cluster.

Redox titrations of the iron-sulphur clusters in fumarate reductase purified from Escherichia coli, monitored by ESR spectroscopy, identified three redox events, similar to those observed in other fumarate reductases and succinate dehydrogenases: Centre 1, a [2Fe-2S] cluster, at g = 2.03, 1.93, appeared on reduction with Em = -20 mV. Centre 3, probably a [3Fe-xS] cluster, at g = 2.02 appeared in the oxidized state with Em = -70 mV. Centre 2 has been observed as an increase in the electron-spin relaxation of Centre 1. It titrates as an n = 1 species with Em = -320 mV, but in our hands did not appear to contribute significant intensity to the g = 2.03, 1.93 signal. It therefore appears to be an additional centre which undergoes spin-spin interaction with Centre 1. The reduction of Centre 2 coincided with the appearance of an extremely broad ESR spectrum, observed at temperatures below 20 K, with features at g = 2.17, 1.9, 1.68. The broad signal was observed in both soluble and membrane-bound preparations. Its midpoint potential was -320 mV. Its integrated intensity was approximately equal to that of Centre 1, if its broad outer wings were taken into account. Consideration of the ESR properties of this signal, together with the amino acid sequence of the frdB subunit of the enzyme, indicates that Centre 2 is a [4Fe-4S] cluster which, in its reduced state, enhances the spin relaxation of the [2Fe-2S] Centre 1.

Electron Spin Resonance Spectroscopy↗
PubMed1986

Reactions of triethylphosphine gold(I) complexes with heme proteins: novel spin-state changes in cytochrome b562, myoglobin, and hemoglobin.

Reactions of bacterial Fe(III) cyt b562, HbO2, met Hb and met Mb with Et3PAuCl and Et3PAuNO3 (and some related complexes) have been investigated by electronic absorption and EPR and NMR spectroscopy. Except for met Hb, which denatured, the products were novel high-spin Fe(III) heme proteins. The reactions of cyt b562 and Mb were reversible. Two distinct kinetic steps were observed in the autoxidation of HbO2 and MbO2. These may involve the liberation of superoxide. Autoxidation of HbO2 occurred more rapidly than that of MbO2. The kinetics of the spin-state change of cyt b562 were too fast to measure by conventional (spectrophotometric) methods. The reaction of Et3PAuCl with HbO2 was not blocked by N-ethylmaleimide. The reactions are discussed in terms of attack by Et3PAu+ on histidine residues in the hydrophobic haem pockets of the proteins.

Animals↗
PubMed1986

Activation and deactivation of the membrane-bound hydrogenase from Desulfovibrio desulfuricans, Norway strain.

The hydrogenase from D. desulfuricans, when isolated in air, had a low activity in the hydrogen-methyl viologen reductase assay, and no activity in the hydrogen-methylene blue reductase assay. The activity increased markedly during incubation under hydrogen. This process is interpreted in terms of conversion of the enzyme from a relatively inactive Unready state to the Active state. Oxidation by dichloro-indophenol caused conversion to a state in which the hydrogen-uptake activity to methyl viologen was preserved, but hydrogen-methylene blue activity was not. This form is termed the Ready state. This behaviour resembles that of the hydrogenase of Desulfovibrio gigas and thus may be a widespread property of this class of hydrogenases. The electron-spin-resonance spectra of the D. desulfuricans enzyme showed the presence of [3Fe-xS] and [4Fe-4S] clusters. Spectra were also observed in the various states of activation of the enzyme. In these respects, the hydrogenase of D. desulfuricans resembles that from D. gigas, although the latter may have an additional iron-sulphur cluster.

2,6-Dichloroindophenol↗
PubMed1986

Activation and active sites of nickel-containing hydrogenases.

Hydrogenases that contain nickel and iron-sulphur clusters also have a regulatory mechanism, by which exposure to oxidants such as oxygen prevents their reaction with hydrogen. Treatment with reducing agents then causes reactivation. In some hydrogenases from Desulfovibrio species, there is evidence that there are at least two different deactivated states, which differ in their rates of reductive reactivation. The membrane-bound hydrogenase of D. desulfuricans, Norway strain, the periplasmic hydrogenase of D. gigas and the membrane-bound hydrogenase of Alcaligenes eutrophus can be isolated in a state (termed "Unready") which requires up to several hours for full activation by hydrogen. By contrast the soluble hydrogenases of D. desulfuricans and A. eutrophus can be reactivated relatively rapidly. In all of these enzymes, with the exception of the latter one, the existence of the activated and deactivated states can be correlated with different ESR-detectable forms of nickel. The possible functions of nickel and [Fe-4S] clusters in catalysis are discussed.

Alcaligenes↗
PubMed1986

The redox properties of the iron-sulphur cluster in hydrogenase from Chromatium vinosum, strain D.

The midpoint potentials of the changes in the electron spin resonance (ESR) spectra in the region of g = 2 in hydrogenase II from Chromatium vinosum were estimated by redox titrations. As the enzyme was progressively reduced, the g = 2.02 signal increased, while the satellite lines at g = 1.98 etc. decreased. At still lower potentials the signal at g = 2.02 decreased. The midpoint potentials of the two processes were estimated to be + 100 mV and - 20 mV, respectively, at pH 8.5. The first potential showed significant pH-dependence. The titration data fitted to n = 1 curves with reasonable reversibility. The enzyme activity showed no significant changes in this potential range. The results are discussed in relation to the interaction of the iron-sulphur cluster with nickel.

Chromatium↗
PubMed1986

A mutant of Escherichia coli fumarate reductase decoupled from electron transport.

The terminal electron-transfer enzyme fumarate reductase of Escherichia coli is a complex iron-sulfur flavoenzyme composed of four nonidentical subunits organized into two domains: FrdA and -B (a membrane-extrinsic catalytic domain) and FrdC and -D (a transmembrane anchor domain). We have identified a mutation within the membrane-intrinsic domain that alters the electron transfer properties of the iron-sulfur and flavin redox centers of the catalytic domain. Functional electron flow from the quinone analog 2,3-dimethyl-1,4-naphthoquinone or from the electron transport chain is impaired. However, the mutant enzyme can be reduced normally by single-electron donors such as the dye benzyl viologen. The mutant phenotype results from a single A----G transition changing His-82, within the second transmembrane alpha-helix of the FrdC anchor sequence, to an arginine. The mutation, physically located within the anchor domain, is manifested by altered catalytic properties, indicating that the intrinsic and extrinsic domains are conformationally connected. These results confirm the important role of the anchor subunits in functional electron transport and have implications for communication between intrinsic and extrinsic domains of membrane proteins.

Base Sequence↗
PubMed1986

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase.

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.

Electron Spin Resonance Spectroscopy↗