F.L. Crane

Studies on the electron transfer system IV. The electron transfer particle

The preparation and properties of an electron transfer particle (ETP) obtained from beef heart mitochondria are described. ETP catalyzes the oxidation of 5.7 μmoles DPNH and 2 μmoles succinate per min per mg at 38°. This specific activity is 3 to 5 times greater than that of the original mitochondrial suspension. These two oxidative reactions are profoundly influenced by 1) concentration of phosphate, 2) the presence of metal complexing agents, and 3) the ionic strength of the medium. Preparations of ETP contain flavin, cytochromes a, b and c1, non-heme iron and copper in constant proportions as well as cytochrome c in variable quantity depending upon the conditions of isolation. Both flavin and hemes are reducible by either substrate and to the same complete extent. Preparations of ETP from which the bulk of cytochrome c has been removed show no requirement for added cytochrome c in the oxidation of DPNH by molecular oxygen, whereas a partial requirement for cytochrome c in the oxidation of succinate is demonstrable under some but not all conditions. The exposure of ETP to deoxycholate leads to a particle which can interact readily with external cytochrome c. Procedures which induce fragmentation of ETP are also described. The significance of the fact of the constant composition of ETP for the dynamics of electron transport is discussed.

Studies on the electron transport system XX. Chemical and physical properties of the coenzyme Q family of compounds

Abstract The chemical and physical properties of four members of a closely related group of compounds are described. These compounds, which function as oxidation-reduction coenzymes in terminal electron transport, have been isolated from beef heart mitochondria, Azotobacter vinelandii and Torula utilis . These compounds are shown to be similar in that each contains the same quinonoid chromophore, probably dialkoxylated. These compounds have different molecular weights, and this difference is at least in part explained by the composition of the mono-unsaturated isoprenoid side chain(s); that is, these compounds differ in the number of such isoprenoid units which occur in the side chain. The formulae of these four compounds which best fit the available data are C 58 H 88 O 4 , C 53 H 80 O 4 , C 48 H 72 O 4 and C 43 H 64 O 4 .

Studies on the electron transport system. XV. Coenzyme Q (Q275) and the succinoxidase activity of the electron transport particle.

Coenzyme Q can be extracted from the electron transport particle and other particles with isooctane. Such extracted particles lose the capacity to oxidize succinate, and this capacity can be restored by addition of coenzyme Q and other lipid supplements. The coenzyme role of Q275 is highly specific. A large number of quinones including vitamin K1 and tocopherol quinone were unable to replace coenzyme Q. No evidence could be found that coenzyme Q is a component of the DPNH chain. On the other hand, cytochrome c is also released in an insoluble form ETP by isooctane, and the addition of cytochrome c fully restores succinoxidase activity of the isooctane-extracted particle. A requirement for both cytochrome c and coenzyme Q in oxidation of succinate can be demonstrated by treating ETP with deoxycholate and isooctane.

Studies on the electron transport system: XVI. Enzymic oxidoreduction reactions of coenzyme Q

Oxidoreduction reactions of coenzyme Q as catalyzed by beef heart mitochondria and derivative particles have been studied. Coenzyme Q is rapidly reduced by substrates, such as pyruvate plus malate, β-hydroxybutyrate, DPNH and succinate. It is also oxidized rapidly by molecular oxygen in the presence of mitochondrial enzymes. The reduction of coenzyme Q by the pyridinoprotein substrates is inhibited by Amytal as well as by antimycin A. The latter compound also inhibits the reduction of coenzyme Q by succinate. The oxidation of reduced coenzyme Q catalyzed by mitochondrial particles is inhibited by cyanide. The possible position of coenzyme Q relative to other members of the electron transport system of beef heart mitochondria is discussed.

Studies on the electron transport system: XVIII. Isolation of coenzyme Q (Q275) from beef heart and beef heart mitochondria

Abstract Two different methods for purification of coenzyme Q from beef heart mitochondria and from beef heart have been described. Selective extraction following saponification or direct extraction of total lipids have been used for initial extraction. The coenzyme Q in these extracts has then been purified by chromatography on Decalso or silicic acid followed by crystallization from ethanol. The coenzyme Q obtained by these two procedures have melting points which range from 49.3 to 50°, E1cm1% at 275 mμ ranges from 162 to 165. All purified preparations have the same Rf when chromatographed on silicone-treated paper and identical visible, ultraviolet and infrared spectra.

Studies on metalloflavoproteins: V. The action of silicomolybdate in the reduction of cytochrome c by aldehyde oxidase

Abstract A silicomolybdate complex has been prepared which will accelerate the reduction of cytochrome c by acetaldehyde in the presence of aldehyde oxidase or xanthine oxidase, but which is without effect upon interaction of these enzymes with other electron acceptors, viz. ferricyanide, 2,6-dichlorophenolindophenol, benzyl viologen, and molecular oxygen. The optimum ratio of molybdenum to silica was found to be ten. Phosphomolybdate will substitute for the silica complex but is not as active in the reduction of cytochrome c. Reduction of either silico- or phosphomolybdate requires the presence of the enzyme, but interaction of the reduced complex and ferricytochrome c is extremely rapid and non-enzymic.

Phospholipase-Induced Release of Cytochrome c from the Electron Transport Particle

Digestion of the electron transport particle with phospholipase A results in the loss of its oxidative capacity. Evidence presented indicates that this is primarily due to the cleavage of the phospholipid-cytochrome c complex within the mitochondria.

Studies on the terminal electron transport system VI. Fragmentation of the electron transport particle with deoxycholate

Abstract At an appropriate ratio of deoxycholate to protein the electron transport particle is fragmented into a red particle similar to the succinic dehydrogenase complex and a green particle which acts as a DPNH oxidase when supplemented with cytochrome c . The red particle contains cytochromes b and c 1 (but not a ) and catalyzes the oxidation of succinate or DPNH by ferricyanide and cytochrome c but not by oxygen. The green particle contains cytochrome b , c 1 and a and catalyzes the oxidation of DPNH (but not succinate) by cytochrome c or by oxygen in presence of cytochrome c . Antimycin inhibits the oxidation of succinate by cytochrome c (catalyzed by the red particle) or oxidation of DPNH by oxygen or cytochrome c (catalyzed by the green particle).

Studies on the electron transport system: XIX. The isolation of coenzyme Q from Azotobacter vinelandii and Torula utilis

Procedures are described for the isolation of 3 crystalline compounds of the coenzyme Q family. One of these compounds was isolated from the nonsaponifiable fraction of cells of Azotobacter vinelandii. Two distinct compounds were isolated from Torula utilis by direct solvent extraction with or without saponification. Chromatographic procedures followed by crystallization techniques are used for the final purification of these three compounds.

Studies on the electron transport system XXVI. Specificity of coenzyme Q and coenzyme Q derivatives

Abstract In succinic dehydrogenase complex there are three alternative pathways by which quinones mediate the oxidation of succinate by cytochrome c or oxygen. The first, mediated by menadione, 2,3-dimethoxy, 5-methyl benzoquinone and the short chain coenzyme Q analogues terminates at external cytochrome c and is antimycin insensitive. The second, mediated only by lipophilic homologues of coenzyme Q (Q 2 , Q 3 , Q 6 , Q 10 ) terminates at external cytochrome c and is antimycin sensitive. The third, mediated by coenzymes Q 2 , Q 3 , Q 6 , Q 10 and in part by Q 0 , Q 1 , plastoquinone and heptyl and heptadecyl coenzyme Q terminates at molecular oxygen, involves bound cytochrome c and is antimycin sensitive. There is a close correlation between the effects of Q analogues on the succinoxidase activity of acetone extracted SDC and the effects on the succinoxidase activity of acetone extracted electron transport particle ETP. This suggests that the reconstituted succinoxidase system of succinic dehydrogenase complex closely resembles that of electron transport particle from which it was derived. Succinic cytochrome c reductase activity of succinic dehydrogenase complex, in principle, is a measure of the segment of the electron transfer chain which has been detached from the cytochrome a terminus of the chain. This segment by virtue of changes induced by the cleavage process shows a new pattern of quinone specificity. Only derivatives of 2,3-dimethoxy, 5-methyl benzoquinone with two or more isoprenoid units in the side chain at carbon 6 are able to serve as mediators in the antimycin sensitive oxidation of succinate by cytochrome c . The antimycin insensitive pathway involving cytochrome c via other quinones involves transfer of electrons from a site in the electron transfer chain prior to the locus for antimycin action.