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D O Hall

Publications and source records attributed to D O Hall.

At least 19 recordsLinked to original sources
PubMed1979

The electron spin relaxation of the electron acceptors of photosystem I reaction centre studied by microwave power saturation.

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electrom transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g=2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g=2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g=2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g=2.07 and 1.86 disappeared and new signals at g=1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B. These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the (4Fe-4S) CENTRes of the electron acceptors A and B resulted in saturation properties which are simular to those of the 2(4Fe-4S) ferredoxin from Clostridium pasteurianum. For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b=1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.

Chloroplasts↗
PubMed1979

Purification of the membrane-bound hydrogenase of Escherichia coli.

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.

Carbon Monoxide↗
PubMed1979

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.

Alcaligenes↗
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↗