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Biomedical subjects

D O Hall

Publications and source records attributed to D O Hall.

At least 55 records · Page 3Linked to original sources
PubMed1978

Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation.

The electron-spin relaxation of iron-sulphur centres in a range of simple proteins (ferredoxin, high-potential iron-sulphur protein and rubredoxin) was investigated by means of the temperature dependence and microwave power saturation of the EPR signal. The proteins containing [2Fe-2S] centres all showed temperature optima higher than those for [4Fe-4S] centres, but the difference between the slowest-relaxing [4Fe-4S] protein (Chromatium high-potential iron-sulphur protein) and the fastest-relaxing [2Fe-2S] protein (Halobacterium halobium ferredoxin) was small. A greater distinction was seen in the power saturation behaviour at low temperature (10--20 K). The behaviour of the signal intensity as a function of microwave power was analyzed in terms of the power for half saturation P 1/2 and the degree of homogeneous/inhomogeneous broadening. The effect of distorting the protein structure by salts, organic solvents and urea was to decrease the electron-spin relaxation rate as shown by a decreased value of P 1/2. The addition of Ni2+ as a paramagnetic perturbing agent caused an increase in the electron-spin relaxation rate of all the proteins, with the exception of adrenal ferredoxin, as shown by an increased P 1/2 and, in a few cases, broadening of the linewidth. Ferricyanide, a commonly used oxidizing agent, has similar effects. These results are discussed in relation to the use of paramagnetic probes to determine whether iron-sulphur centres are near to a membrane surface. Spin-spin interactions between two paramagnetic centres in a protein molecule such as a 2[4Fe-4S] ferredoxin, lead to more rapid electron-spin relaxation. This method was used to detect a spin-spin interaction between molybdenum V and centre Fe-SI in xanthine oxidase.

Chromatium↗
PubMed1978

Subcellular localisation and identification of superoxide dismutase in the leaves of higher plants.

1. The subcellular location of superoxide dismutase in the leaves of spinach and other C3 plants has been investigated. 2. Most activity appeared to be located within chloroplasts. These organelles contain a cyanide-sensitive (copper-zinc) superoxide dismutase, most of which is located in the stroma although some is bound to the thylakoids. 3. Intact chloroplast fractions also contain a cyanide-insensitive (manganese) superoxide dismutase, but this activity is located on the outside of the chloroplasts and may be adsorbed onto them during isolation. 4. Leaf mitochondrial fractions contain only a small percentage of total leaf superoxide dismutase activity, but there is more than can be accounted for by contamination with chloroplasts. 5. Mitochondria contain both a cyanide-sensitive dismutase, apparently located in the intermembrane space, and a cyanide-insensitive activity, apparently located in the matrix. 6. The microsomal fraction contains no superoxide dismutase activity.

Chloroplasts↗
PubMed1978

Circular dichroism and magnetic circular dichroism of iron-sulfur proteins.

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra are reported for the 2-Fe ferredoxins from Pseudomonas putida and Spirulina maxima, Chromatium HIPIP, the 4-Fe ferredoxin from Bacillus stearothermophilus, and the 8-Fe ferredoxin from Clostridium pasteurianum. The spectral range spans the near-infrared, visible, and near ultraviolet. In all cases except oxidized 2-Fe ferredoxins, electronic absorption is observed continuously from less than 5000 cm-1 to above 30,000 cm-1. The CD spectra of the two 2-Fe ferredoxins are similar. In contrast, the CD of the 4-Fe and 8-Fe proteins, for a given 4-Fe cluster oxidation level, varies considerable with protein. MCD is less sensitive to protein environment than is CD. In the 2-Fe proteins, MCD at 5 T is appreciably smaller than the CD; in the 4-Fe and 8-Fe proteins, MCD and CD are comparable in magnitude. Both CD and MCD are more highly structured than the corresponding absorption spectra. The CD and MCD spectra reported provide a broader base than heretofore available for the characterization of iron-sulfur proteins containing 2-Fe and 4-Fe clusters and for the evaluation of electronic structural models for these clusters.

Bacteria↗
PubMed1978

Superoxide dismutase and catalase in the protection of the proton-donating systems of nitrogen fixation in the blue-green alga Anabaena cylindrica.

1. Superoxide dismutase activity was present in the heterocysts and vegetative cells of Anabaena cylindrica, but was always lower in the heterocysts. 2. No qualitative differences were found in the superoxide dismutase from the two cellular types. 3. Catalase activity was also present in both cellular types. 4. Most of the NADP reductase activity, as assayed with menadione or ferredoxin as electron acceptor, was localized within the heterocysts. 5. Studies on H2 consumption showed that most of the hydrogenase activity was associated with the heterocysts. 6. The results are discussed in terms of the postulate that superoxide dismutase and catalase are involved in the protection of the proton-donating systems participating in N2 fixation and H2 metabolism of heterocysts.

Catalase↗
PubMed1978

Oxidation-reduction studies of the Mo-(2Fe-2S) protein from Desulfovibrio gigas.

Potentiometric titration followed by e.p.r. measurements were used to determine the midpoint reduction potentials of the redox centres of a molybdenum-containing iron-sulphur protein previously isolated from Desulfovibrio gigas, a sulphate-reducing bacterium (Moura, Xavier, Bruschi, Le Gall, Hall & Cammack (1976) Biochem. Biophys. Res. Commun. 728 782-789; Moura, Xavier, Bruschi, Le Gall & Cabral (1977) J. Less Common Metals 54, 555-562). The iron-sulphur centres could readily be distinguished into three types by means of g values, temperature effect, oxidation-reduction potential values and reduction rates. The type-I Fe-S centres are observed at 77 K. They show mid-point potential values of -260mV (Fe-S type IA) and -440 mV (Fe-S type IB). Centres of types IA and IB appear to have similar spectra at 77 K and 24 K. The Fe-S type-II centres are only observed below 65 K and have a midpoint potential of -28mV. Long equilibration times (30 min) with dye mediators under reducing conditions were necessary to observe the very slow equilibrating molybdenum signals. The potential values associated with this signal were estimated to be approx. -415 mV for Mo(VI)/Mo(V) and-530mV for Mo(V)/Mo(IV).

Desulfovibrio↗
PubMed1978

Hydrogen evolution by chloroplast-hydrogenase systems: improvements and additional observations.

An in vitro system containing isolated chloroplasts, ferredoxin and bacterial hydrogenase on illumination evolves H2 and O2 from water. Maximum rate of hydrogen production so far achieved is two litres H2 per g. chlorophyll per h. The rate of H2 evolution per mg chlorophyll is dependent on concentrations of chlorophyll and ferredoxin in the reaction mixture. The rates as well as duration of H2 production are enhanced by the presence of oxygen scavengers and bovine serum albumin in the system. Hydrogenases and ferredoxins vary in their degree of cross reactivity in the chloroplast system; with some hydrogenases the H2 evolution rates were increased by the presence of additional biological electron carriers. Attempts to couple algal hydrogenases to the chloroplasts system have not succeeded so far.

Bacteria↗
PubMed1978

Cluster characterization in iron-sulfur proteins by magnetic circular dichroism.

We report magnetic circular dichroism (MCD) spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothermophilus ferredoxin and Clostridium pasteurianum ferredoxin. The MCD is found to vary significantly with cluster oxidation state but is relatively insensitive to the nature of the protein. The spectra obtained are compared with the corresponding spectra of iron-sulfur proteins containing 2-Fe clusters. It is concluded that MCD is useful for the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins and is superior for this purpose to absorption and natural circular dichroism spectroscopy.

Chromatium↗
PubMed1978

Complete amino acid sequence of Halobacterium halobium ferredoxin containing an Nepsilon-acetyllysine residue.

1. The complete amino acid sequence of the 2Fe-2S ferredoxin from Halobacterium halobium was determined to be: (formula see text):2. The apoferredoxin chain consists of 128 amino acid residues and has a molecular weight of 14,330. 3. There are only four cysteines in this ferredoxin molecule; they should be involved in the binding of the two iron atoms at the active center. Ther relative positions of these cysteines are similar to those of the cysteines in chloroplast ferredoxins. 4. There is a high degree of homology between H. halobium ferredoxin and chloroplast ferredoxins, though the latter molecules contain only about 98 amino acid residues. 5. H. halobium ferredoxin contains a single residue of Nepsilon-acetyllysine.

Amino Acid Sequence↗
PubMed1977

The low temperature magnetic circular dichroism spectra of iron-sulphur proteins. I. Oxidised rubredoxin.

Variable temperature magnetic circular dichroism spectra have been measured on oxidised Clostridium pasteurianum rubredoxin. Evidence has been obtained for the presence of two one-electron charge-transfer transitions, sulphur to ferric ion, in the region 15 000 to 28 000 cm-1. The first moment of the lower energy band is consistent with it being the orbital transition t1 non-bonding sulphur orbital, to the 2 e ferric d-orbital. The magnitude of the spin-orbit coupling constant in the lower excited state has been determined and shown to be small compared with the axial distortion. The splitting of the low energy band observed in the absorption spectrum can therefore be equated directly with the axial distortion of the lowest excited charge-transfer state. Finally, the potential utility of making saturation experiments at very low temperatures has been examined.

Circular Dichroism↗
PubMed1977

The low temperature magnetic circular dichroism spectra of iron-sulphur proteins. II. Two-iron ferredoxins.

Variable temperature magnetic circular dichroism (MCD) spectra of a number of two-iron ferredoxins have been measured. The spectra of fully oxidised spinach and Spirulina maxima ferredoxin are independent of temperature between room temperature and 18 K, showing that no contribution to the room temperature MCD spectrum arises from the small population of low-lying excited states originating from the exchange coupling. However, the low temperature MCD spectra of the half-reduced proteins spinach and Spirulina maxima ferredoxin and adrenodoxin are all reasonably intense and temperature dependent. An interpretation of the spectrum of the charge-transfer region is suggested by starting with the assignments previously obtained from rubredoxin.

Circular Dichroism↗
PubMed1977

Spectroscopic studies of the oxidation-reduction properties of three forms of ferredoxin from Desulphovibrio gigas.

Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.

Binding Sites↗
PubMed1977

Comparative immunochemistry of bacterial, algal and plant ferredoxins.

1. Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus, the 2 [4Fe-4S] ferredoxin from Clostridium pasteurianum, and the 2Fe-2S ferredoxins from the blue-green algia Spirulina maxima, the green alga Scenedesmus obliquus, and the higher plant Beta vulgaris. The antibodies were tested for immunoprecipitation activity with seven bacterial, twelve blue-green algal, six eukaryotic algal and six higher plant ferredoxins. 2. Antibodies to the bacterial ferredoxins reacted to a significant extent only with their homologous proteins. On the other hand, antibodies to the plant and algal ferredoxins showed cross-reaction with other ferredoxins. There was a correlation between the degrees of immunoprecipitation and the similarity in amino acid sequences. These results suggest that the method can be used as a marker in taxonomic studies. 3. The interaction of the antibodies with the five native ferredoxins was compared with the reactions with their apoproteins. In each case the degree of interaction was different. This behaviour was interpreted as due to an influence of tertiary structure on the antibody-antigen interaction.

Amino Acid Sequence↗