Search PubMed⌕ Search

Biomedical subjects

B A Ackrell

Publications and source records attributed to B A Ackrell.

At least 55 records · Page 3Linked to original sources
PubMed1985

Detection of a tetranuclear iron-sulfur center in fumarate reductase from Escherichia coli by electron paramagnetic resonance spectroscopy.

Soluble fumarate reductase and fumarate reductase complex from Escherichia coli have been investigated by electron paramagnetic resonance spectroscopy. Both succinate- and dithionite-reduced samples show signals associated with a [2Fe-2S]1+ cluster that account maximally for slightly more than one spin/molecule. In addition, at temperatures below 20 K, dithionite-reduced samples exhibit broad and complex features, to high and low field of the [2Fe-2S]1+ signal, that are attributable to a spin coupled [4Fe-4S]1+ cluster. Preliminary attempts to quantify the signals indicate that the [4Fe-4S] cluster is present in an approximate 1:1 stoichiometry with the [2Fe-2S] cluster. The observed enhancement of the spin relaxation of the [2Fe-2S]1+ cluster on dithionite reduction is attributed to spin-spin interaction between the S = 1/2, reduced tetranuclear and binuclear clusters.

Dithionite↗
PubMed1985

Electron paramagnetic resonance studies of mammalian succinate dehydrogenase. Detection of the tetranuclear cluster S2.

Electron paramagnetic resonance studies of Complex II from the mitochondrial respiratory chain and soluble preparations of succinate dehydrogenase have, for the first time, identified a signal arising from a [4Fe-4S]1+ cluster, S2, in dithionite-reduced samples. Redox titrations, monitored by electron paramagnetic resonance spectroscopy demonstrate that this signal appears at the same midpoint potential as the enhancement of the spin relaxation properties of the [2Fe-2S]1+ center, S1, in both Complex II and reconstitutively active soluble enzyme. The results complement recent magnetic circular dichroism studies of succinate dehydrogenase (Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, B. A. C., and Kearney, E. B. (1985) J. Biol. Chem. 260, 7368-7378) which assigned cluster S2 as a [4Fe-4S]2+,1+ center and provide evidence for spin interaction between the paramagnetic reduced forms of centers S1 and S2.

Animals↗
PubMed1985

Magnetic circular dichroism studies of succinate dehydrogenase. Evidence for [2Fe-2S], [3Fe-xS], and [4Fe-4S] centers in reconstitutively active enzyme.

Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.

Animals↗
PubMed1984

Effect of iron deficiency on succinate- and NADH-ubiquinone oxidoreductases in skeletal muscle mitochondria.

The effects of iron deficiency on the NADH- and succinate-oxidizing complexes of rat skeletal muscle mitochondria have been investigated. Both systems were similarly affected: activities were about 30% of normal in dehydrogenase, ubiquinone reductase, and oxidase assays, and similar reductions in the concentration of their respective flavin prosthetic groups were also evident in the iron-deficient membranes. Thus, the turnover numbers of the two enzymes were unchanged in iron deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed similarly reduced levels of those peptide components of Complexes I and II that could be unequivocally distinguished. Soluble beef heart succinate dehydrogenase added to alkaline-treated rat skeletal muscle mitochondrial membranes attached to binding sites exposed by the treatment, forming a hybrid complex indistinguishable from the original skeletal muscle complex, with restoration of succinoxidase and succinate-ubiquinone reductase activities to the levels observed in the original rat membranes. Iron-deficient particles behaved like the normal in these tests. No unfilled binding sites for the enzyme could be detected prior to alkaline treatment. The data are interpreted as indicating that the lower activities of these two respiratory complexes in iron deficiency are due to lower content of the enzymes rather than to the presence of impaired enzymes in the membrane, that only fully competent complexes are present in these membranes, and that iron-deficient complexes are either not assembled or are lost after assembly.

Animals↗
PubMed1984

Iron-sulfur cluster 3 of beef heart succinate-ubiquinone oxidoreductase is a 3-iron cluster.

From a study of the magnetic field dependence of the linear electric field effect (LEFE) in EPR spectroscopy, we demonstrate that iron-sulfur cluster 3 in air-oxidized, beef heart succinate-ubiquinone oxidoreductase (Complex II) is a 3-iron cluster. This suggests that cluster 3 may arise by oxidative degradation from a 4-iron cluster originally present in the enzyme and may be reconverted back into a 4-iron cluster under reducing conditions. Linear electric field effect studies of succinate-reduced Complex II are in accord with the view that cluster 1 is a 2-iron cluster.

Animals↗
PubMed1981

Reaction site of carboxanilides and of thenoyltrifluoroacetone in complex II.

Oxathiin carboxanilides are systemic fungicides that inhibit the oxidation of succinate by interrupting electron transport between succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1] and coenzyme Q. Kinetic and electron paramagnetic resonance studies have established that the specific binding site of carboxanilides and of thenoyltrifluoroacetone responsible for the inhibition is the same. Although the binding of carboxanilides to membrane preparations of the dehydrogenase is very tight (Ki = 0.01-0.1 microM), it is noncovalent. Identification of the membrane component(s) to which specific binding occurs has therefore required the introduction of a photoaffinity label onto the carboxanilide molecule. By using [G-3H]3'-azido-5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide, it was found, in accord with earlier data with other carboxanilides, that unresolved complex II specifically binds about 0.6 mol of the inhibitor per mol of succinate dehydrogenase in equilibrium dialysis experiments. The resolved components of the complex, succinate dehydrogenase and the two binding peptides CII-3 and CII-4, failed to bind the inhibitor; however, when these were recombined with reconstitution of coenzyme Q reductase activity, the initial binding titer was restored. Azidocarboxanilide-inhibited complex II was irradiated to generate covalent linkages with the binding site, and the components of the complex were separated on polyacrylamide gel. Most of the specifically bound inhibitor was found in the low molecular weight binding peptides and phospholipids.

Anilides↗
PubMed1977

Isolation of reconstitutively active succinate dehydrogenase in highly purified state.

Existing procedures for the isolation of mammalian succinate dehydrogenase yield preparations of high purity or retain reconstitution activity, but not both. A new procedure is described for the isolation in good yield of virtually homogeneous preparations with full reconstitution activity, and retaining iron-sulfur center 3 and the "low Km" reaction site of ferricyanide. On reincorporation of the soluble enzyme into alkali-treated membranes, the same high turnover number (approximately 21,000/min at 38 degrees) is obtained in catalytic assays as with intact inner membrane preparations.

Animals↗
PubMed1977

Effect of membrane environment on succinate dehydrogenase activity.

The turnover number of succinate dehydrogenase from mammalian heart determined by the spectrophotometric phenazine methosulfate assay, after complete activation, is approximately 21,000 mol of succinate oxidized/min/mol of histidyl flavin at 38 degrees in relatively intact inner membrane preparations and mitochondria. Reconstitutively active soluble preparations, extracted anaerobically in the presence of succinate from inner membrane preparations show turnover numbers of 11,500 to 14,500 and a significantly lower apparent Km for phenazine methosulfate than the parent particles. The decline of both the turnover number and of the Km occurs during the brief period when the enzyme is detached from the membrane. The observed values represent the activities in the soluble extract of both the reconstitutively active and reconstitutively inactive enzyme. The latter may be from 10 to 40% even in the most carefully prepared enzyme; it has a lower turnover number in the phenazine methosulfate assay than the average for the solution and is devoid of catalytic activity in the "low Km" ferricyanide assay (Vinogradov, A. D., Ackrell, B.A.C., and Singer, T.P. (1975) Biochem. Biophys. Res. Commun. 67, 803-809). The reconstitutively active form of the soluble enzyme has a turnover number of at least 15,000 and an equal activity in the low Km ferricyamide assay. When recombined with the membrane the total activity of the enzyme is increased by over 60% and it regains the original turnover number, Km for phenazine methosulfate, and sensitivity of the phenazine methosulfate reductase activity to thenoyltrifluoroacetone, carboxamides, and cyanide. It appears, therefore, that the membrane environment or some component of it exerts a positive modulating influence on the enzyme even in the fully activated state. In certain particulate sources (Keilin-Hartree preparations, Complex II) the enzyme shows lower turnover numbers (11,000 to 12,500) than in more intact inner membranes. This seems to be due to inactivation in the course of preparation and, in the case of Complex II, in part also to loss of the normal membrane environment or of a membrane component, possibly Q-10, during isolation.

Animals↗