Y. Hatefi

Studies on the mechanism of oxidative phosphorylation III. Phosphorylating particle types from beef heart

Crude mitochondrial suspensions, prepared on a large scale from beef heart, have been fractionated by centrifugation in a buffered medium. Two principal fractions, heavy and light, have been isolated, and some of their properties with respect to the oxidation of citric acid cycle substrates and the accompanying phosphorylation have been studied. The heavy fraction, which resembles intact mitochondria much more closely than the light, catalyzes the oxidation of these substrates very efficiently with P/O ratios quite close to the assumed theoretical values. These preparations can be conveniently stored in the deep-freeze for long periods of time without considerable loss of activity.

Studies on the electron transport system. XXXII. Respiratory control in beef heart mitochondria

Abstract Respiratory control has been studied with preparations of beef heart mitochondria. The oxidation of pyridinoprotein substrates is strongly regulated by a respiratory control mechanism, while the oxidation of succinate is not. A small degree of control can be demonstrated for the oxidation of succinate, which is abolished upon addition of Amytal to the reaction mixture. These as well as other observations indicate the possibility that respiratory control is associated only with the first phosphorylation site in the DPNH oxidase system of beef heart mitochondria.

Studies on the electron transport system. XXX. DPNH-cytochrome c reductase I.

Abstract A highly active DPNH-cytochrome c reductase has been isolated from beef-heart mitochondria. The best preparations of the enzyme catalyze the reduction by DPNH of approx. 50–60 μmoles cytochrome c /min/mg protein at 38°. The enzymic activity is completely inhibited by Amytal, p -chloromercuriphenyl sulfonate, antimycin A, SN-5949 or 2-nonyl-4-hydroxyquinoline-N-oxide, and is stimulated by EDTA. The preparation contains DPNH flavoprotein, cytochromes b and c 1 , Coenzyme Q and non-heme iron and is essentially free of succinic-cytochrome c reductase as well as cytochrome oxidase activity.

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 the electron transport system: XVII. Effects of adenosine diphosphate and inorganic phosphate on the steady-state oxidereduction level of coenzyme Q

Abstract The oxidoreduction reactions of coenzyme Q have been studied under steady-state conditions. Inorganic phosphate affects a marked reduction of coenzyme Q, and ADP reverses this reduction. Other anions such as sulfate, nitrate, borate, pyrophosphate and chloride, cannot duplicate the effect of phosphate, while arsenate can. The reduction induced by arsenate is not, however, reversed by ADP. The studies with phosphate, arsenate and ADP have been extended to DPN and exactly analogous results obtained. The phosphate-induced reduction of coenzyme Q is inhibited by both Amytal and antimycin A. These studies indicate that coenzyme Q, like DPN, acts as an electron carrier during the process of oxidative phosphorylation. In addition, evidence is presented that a high-energy bond is formed first with phosphate rather than with ADP. This is the first demonstration of the order in which phosphate and ADP participate in oxidative phosphorylation. Thus, in the terminal reaction of oxidative phosphorylation. ATP is formed by the transfer of the high-energy phosphoric group to ADP. The implication of these results in the mechanism of oxidative phosphorylation is discussed.

Studies on the electron transport system. XXIII. Coenzyme Q oxidase.

The preparation and properties of a submitochondrial enzyme system containing predominantly cytochrome a have been described. This system is capable of catalyzing the oxidation of reduced coenzyme Q by molecular oxygen. This particulate enzyme requires the presence of both cytochrome c and a lipoproteinof mitochondrial origin in order to catalyze the above reaction. The results presented in this communication suggest that coenzyme Q is situated in the aforementioned lipoprotein between cytochromes b and c.

Studies on the mechanism of oxidative phosphorylation: IV. Pyridine nucleotide binding and its relation to activity in heart mitochondria

Abstract The oxidative phosphorylation activity of beef-heart particles with respect to the substrates of pyridino-protein enzymes is directly proportional to the amount of particle-bound pyridine nucleotides. The pyridine nucleotide content of the different particle types decreases in the following order: heavy particles, light particles, PETP and ETP. When particles are incubated aerobically, the pyridine nucleotides are released and rapidly destroyed, and simultaneously the capacity for pyridine nucleotide-linked oxidation declines. This loss of activity can be restored completely by addition of DPN and EDTA to the assay. After long-term aerobic incubations of mitochondria, the oxidation induced by DPN and EDTA is largely uncoupled. However, further addition of bovine serum albumin during the assay leads to restoration of a considerable proportion of the lost capacity for oxidative phosphorylation. The inactivation due to aerobic incubation can be prevented under anaerobic conditions or by addition to the incubation medium of substrates, EDTA, Mn ++ or Mg ++ . These effects, particularly in the case of protection by EDTA, are due to inhibition of the release of particle-bound pyridine nucleotides. The implications of these results with respect to the possible factors involved in binding of pyridine nucleotides are discussed.