David O. Hall

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.

The stoichiometry (ATP-2e- ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts.

Abstract 1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2 e − ) and photosynthetic control ratios. 2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation. 3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH 4 Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II. 4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained. 5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “ in vivo ” to allow CO 2 fixation to proceed.

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.

Low-temperature magnetic circular dichroism evidence for the conversion of four-iron-sulphur clusters in a ferredoxin from Clostridium pasteurianum into three-iron-sulphur clusters

Oxidation of the 8Fe ferredoxin from Clostridium pasteurianum with potassium ferricyanide, followed by purification on Sephadex G-25 and DE-23 cellulose columns, gives a protein with an intense EPR signal at g 2.01. The low-temperature magnetic circular dichroism (MCD) spectra of this species are different from those of the oxidized high-potential iron protein from Chromatium but identical with the spectra of ferredoxin II from Desulphovibrio gigas. On reduction of the ferricyanide-treated ferredoxin with sodium dithionite only a weak EPR signal with g factors of 2.05, 1.94 and 1.89 is obtained. The low-temperature MCD spectra are strongly temperature dependent with a form similar to those of dithionite-reduced D. gigas ferredoxin II. The MCD magnetization curves are dominated by a species with ground-state effective g factors of gi 8.0 and g⊥ 0.0, which are also similar to those determined recently by low-temperature MCD spectroscopy for D. gigas ferredoxin II. The MCD characteristics are quite different from those of dithionite-reduced ferredoxin from Cl. pasteurianum, untreated with ferricyanide. This establishes the close similarity of the iron-sulphur clusters in ferricyanide-treated Cl. pasteurianum ferredoxin and in D. gigas ferredoxin II. The latter is known to contain a single 3Fe centre, similar to that observed in ferredoxin I from Azotobacter vinelandii by X-ray crystallography. Therefore, it is concluded that the [4Fe-4S] clusters of Cl. pasteurianum ferredoxin are converted to 3Fe clusters on oxidation with ferricyanide.

Soluble & membrane-bound superoxide dismutases in a blue-green alga (spirulina) and spinach

Abstract Soluble extracts of Spirulina contained three cyanide-insensitive superoxide dismutase activities. The major isoenzyme was purified and shown to contain iron. Soluble extracts of spinach leaves contained two distinct cyanide-sensitive ( Cu Zn ) dismutase activities one of which occurred in the stroma of isolated chloroplasts. A cyanide-insensitive dismutase activity was associated with chloroplast lamellar membranes: this was solubilised and partially purified. By subfractionation of lamellae, a dismutase activity was shown to be bound to particles with a chlorophyll a b ratio of about 2 (photosystem II). These results are discussed in terms of the possible in vivo functions of the different enzymes.

The superoxide dismutase activity of various photosynthetic organisms measured by a new and rapid assay technique

Reactions dependent on the presence of 02 are inhibited by addition of superoxide dismutase [1 ] and this observation has led to the development of a number of assays for measuring the activity of this enzyme. The assays usually consist of (i) a system which generates O~, e.g. mixtures of xanthine and xanthine oxidase [5], NADH and phenazine methosulphate [6] or an illuminated mixture of riboflavirr and methionine [7] together with (ii) a compound which reacts with O~ such as cytochrome c [5], nitro-blue tetrazolium [7], adrenaline [8], pyrogallol [9] or hydroxylamine [10]. The ability of an added cell extract to inhibit the latter reactions is taken as a measure of its euperoxide dismutase activity. However, inhibition could also be attributed to prevention of the formation of O~ in these complex reaction mixtures (e.g. [11] ). Further, it is very difficult to study the effects of metabolites on superoxide dismutase activity, because of the possibility of interference with different stages of these assay systems [1 ]. In the present paper a rapid and simple assay for superoxide dismutase is described, which utilizes the ability of these enzymes to inhibit reduction of nitroblue tetrazolium by K÷O~ added directly as a solution in dimethylsulphoxide [ 12,13 ].

A stable and easily extractable plant-type ferredoxin from the blue-green alga Spirulina maxima

The 2Fe + 2S plant-type ferredoxin from the blue-green alga Spirulina, maxima can be easily extracted in high yield −35 mg ferredoxin/100 g dried cells. The cells can be stored as a dried powder which is readily obtainable. The purified ferredoxin is very stable even aerobically at room temperature. It is much more stable than the five other plant-type ferredoxins with which it was compared. The biological activity of all ferredoxins was comparable.

Spin lattice relaxation and exchange interaction in a 2-iron, 2-sulphur protein

A two-iron-two-sulphur non-haem iron protein, the ferredoxin from Spirulina maxima, has been studied by means of electron paramagnetic resonance (EPR) in the range where the spectrum loses resolution with increasing temperature. The spin-lattice relaxation times were deduced from linewidths measured by spectral simulation and their variation as a function of temperature is interpreted in terms of an Orbach mechanism. On this basis, the exchange integral between the two iron atoms, assuming as antiferromagnetic interaction between them, is estimated to be - 83 cm-1.

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.

The iron electron-nuclear double resonance (ENDOR) of 4-Fe clusters in iron-sulfur proteins from Chromatium and Clostridium pasteurianum

Iron electron-nuclear double resonance (ENDOR) measurements were made of the 4-Fe clusters in oxidized Chromatium high-potential iron-sulfur protein, dithionite-reduced high-potential iron-sulfur protein in 80% dimethylsulphoxide, fully reduced Clostridium pasteurianum ferredoxin in aqueous solution and in 80% dimethylsulfoxide. The hyperfine couplings determined show that: i) the electron distribution in each case is nearly symmetric; ii) there are two types of iron in oxidized high potential iron-sulfur protein; iii) only one type of iron is observed in each fully reduced 4-Fe cluster; iv) the data also suggest a greater electron delocalization onto the ligands as compared to the 2-Fe ferredoxins.