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A L Moore

Publications and source records attributed to A L Moore.

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Characterization of iron-sulphur centres of plant mitochondria by microwave power saturation.

The electron spin relaxation of iron-sulphur centres and ubisemiquinones of plant mitochondria was studied by microwave power saturation of the respective EPR signals. In the microwave power saturation technique, the experimental saturation data were fitted by a least-squares procedure to a saturation function which is characterized by the power for half-saturation (P1/2) and the inhomogeneity parameter (b). Since the theoretical saturation curves were based on a one-electron spin system, it became possible to differentiate between EPR signals of iron-sulphur centres which have similar g values but different P1/2 values. If the difference in the P1/2 values of the overlapped components was small, no significant deviation from these theoretical saturation curves was observed, as shown for the overlapped signals of centre S-3 and the Ruzicka centre of mung bean mitochondria. By contrast, the microwave power saturation data for the g = 1.93 signal (17--26 K) of Arum maculatum submitochondrial particles reduced by succinate could not be fitted using one-electron saturation curves. Reduction by NADH resulted in a stronger deviation. Since the iron-sulphur centres of Complex I were present only in an unusually low concentration in A. maculatum mitochondria, it was proposed that an iron-sulphur centre of the external NADH dehydrogenase contributes to the spectrum of centre S-1. For mung bean mitochondria, the g = 1.93 signal below 20 K could be attributed mainly to centre N-2. The microwave power saturation technique was also suitable for detecting magnetic interactions between paramagnetic centres. From the saturation data of the complex spectrum attributable to centre S-3 and an interacting ubisemiquinone pair in mung bean mitochondria (oxidized state) followed that centre S-3 has a faster electron spin relaxation than the ubisemiquinone molecules. It is noteworthy that the differences in the relaxation rates were maintained despite the interaction between centre S-3 and the ubisemiquinones. Furthermore, a relaxation enhancement was observed for centre S-1 of A. maculatum submitochondrial particles upon reduction of centre S-2 by dithionite. This indicated a magnetic interaction between centres S-1 and S-2.

Electron Spin Resonance Spectroscopy

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

Studies on the mechanism of inhibition of redox enzymes by substituted hydroxamic acids.

Substituted primary hydroxamic acids were found to inhibit the catalytic activity of a number of redox enzymes. The inhibition was not related to the nature of the metal-active site of the enzyme nor to the nature of the oxygen-containing substrate. Two easily available enzymes, mushroom tyrosinase (monophenol,dihydroyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which were potently inhibited by hydroxamic acids, were chosen for more detailed study. A kinetic analysis of the inhibitory effects on the partially purified tyrosinase of mushroom (Agaricus bispora) revealed that inhibition was reversible and competiitive with respect to reducing substrate concentration, but was not competitive with respect to molecular oxygen concentration. A spectrophotometric and EPR study of the binding of salicylhydroxamic acid to horseradish peroxidase revealed that his hydroxamic acid was bound to the enzyme in the same manner as a typical substrate, hydroquinone. Spectroscopic and thermodynamic measurements of the binding reactions suggested that this binding site is close, to but, not directly onto, the heme group of the enzyme. From these results it is concluded that the mode of inhibition of hydroxamic acid need not be, as generally supposed, by metal chelation, and mechanisms involving either hydrogen bonding at the reducing substrate binding site or the formation of a charge transfer complex between hydroxamic acid and an electron-accepting group in the enzyme are considered to be more feasible. The relevance of these findings to deductions on the nature of other hydroxamic acid-inhibitable systems is discussed.

Basidiomycota

EPR studies of higher plant mitochondria. I Ubisemiquinone and its relation to alternative respiratory oxidations.

An EPR investigation of the region of the higher plant respiratory chain involving ubiquinone and Center S-3 of succinate dehydrogenase is reported. At temperatures close to those of liquid helium, first derivative spectra corresponding to Center S-3 (gmax = 2.017) and a signal split around g = 2.00 (major features of peaks and troughs at g values of 2.045, 2.03, 1.985, 1.97 and 1.96) were observed in mung bean (Phaseolus aureus), Arum maculatum spadix, Sauromatum guttatum spadix and tulip bulb (Tulipa gesnerana) mitochondria. The split signal was small or absent in potato tuber and Symplocarpus foetidus spadix mitochondria. The redox behavior of these signals in mung bean mitochondria in a variety of respiratory steady-state conditions suggested that the components giving rise to them were an integral part of the respiratory chain and were located on the substrate side of coupling Site II. The split signal could be removed by addition of hydroxamic acids in all tissues tested, although the Ks of this effect was an order of magnitude higher than the Ki of inhibition of the alternative respiratory pathway in mung bean and Sauromatum guttatum spadix mitochondria. The results are discussed in relation to the current ideas on the ordering of components in the region around the classical Site II of the respiratory chain and in relation to the location of the alternative respiratory oxidase pathway of higher plants.

Cyanides

The effect of calcium on the respiratory responses of mung bean mitochondria.

Purified mung bean hypocotyl mitochondria were examined for their capacity to carry out respiration-dependent accumulation of calcium. The addition of 0.1-1.0 mM calcium to mung bean mitochondria supplemented with succinate gave no stimulation of state 4 respiration even in the presence of inorganic phosphate and the ionophoretic antibiotic A-23187. Even at high calcium concentrations, no transient changes in the respiratory activity occurred and subsequent addition of ADP initiated a further state 3 response. Although the additions of calcium resulted in a rapid H+ ejection, it was insensitive to lanthanum and uncoupling agents. Similarly, additions of calcium failed to initiate any transient changes in the oxidation-reduction states of either pyridine nucleotides or cytochrome b. Direct spectrophotometric recordings of absorbance changes of murexide revealed no respiration-linked calcium transport. It is proposed that although mung bean mitochondria possess a respiration-linked electrochemical potential gradient it would appear that this potential cannot be expressed as calcium transport even at high ion concentrations, probably due to a low calcium membrane permeability.

Anaerobiosis

The effects of bathophenanthroline, bathophenanthrolinesulphonate and 2-thenoyltrifluoroacetone on mung-bean mitochondria and submitochondrial particles.

The effects of bathophenanthroline, bathophenanthrolinesulphonate and 2-thenoyltrifluoroacetone on mung-bean mitochondria and submitochondrial particles were investigated. A variety of inhibitory effects on the oxidations of NADH, succinate and malate were observed. The results are discussed in relation to sites of inhibition and their relation to the effects on mammalian mitochondria.

Malates