1. Pigeon heart mitochondria produce H(2)O(2) at a maximal rate of about 20nmol/min per mg of protein. 2. Succinate-glutamate and malate-glutamate are substrates which are able to support maximal H(2)O(2) production rates. With malate-glutamate, H(2)O(2) formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates. 3. Antimycin A exerts a very pronounced effect in enhancing H(2)O(2) production in pigeon heart mitochondria; 0.26nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H(2)O(2) formation. 4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H(2)O(2) formation. 5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H(2)O(2) production is linearly related to the incorporated ubiquinone. 6. The generation of H(2)O(2) by pigeon heart mitochondria in the presence of succinate-glutamate and in metabolic state 4 has an optimum pH value of 7.5. In states 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values. 7. With increase of the partial pressure of O(2) to the hyperbaric region the formation of H(2)O(2) is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in state 4, an increase in the pO(2) up to 1.97MPa (19.5atm) increases H(2)O(2) formation 10-15-fold. Similar pO(2) profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O(2) was observed up to 1.97MPa (19.5atm). By increasing the pO(2) to 1.97MPa (19.5atm), H(2)O(2) formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic state 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent K(m) of about 71kPa (0.7-0.8atm) and a V(max.) of 12 and 20nmol of H(2)O(2)/min per mg of protein. 8. It is postulated that in addition to the well-known flavin reaction, formation of H(2)O(2) may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome b level.

The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen.

Metabolism and function of coenzyme Q

1) Introduction a) Aging is the process of getting older b) Gerontology is the study of aging c) A lot of what we know about aging is associated with biology d) Why study aging? i) Increase in the number of people age 65 and over ii) 1970: 10% of population is elderly; 2030 projected that 21.2% iii) Population pyramids (usually only a few at top and many at bottom, now many are getting older) iv) Why are more people getting older? e) Aging upsets ability to maintain homeostasis

The biology of aging.

1
Submitochondrial particles were prepared from beef-heart mitochondria by sonication in the presence of EDTA. The particles were lyophilized and repeatedly extracted with pentane until no ubiquinone was found in the extract. Treatment of the ubiquinone-depleted particles with pentane containing a suitable concentration of ubiquinone (ubiquinone-50) and subsequent quick washing with ubiquinone-free pentane resulted in a “re-incorporation” of ubiquinone in an amount similar to that present in the original particles (3–6 nmoles/mg protein).

2
The ubiquinone-depleted particles exhibited very low or no succinate and NADH oxidase activities, which were restored upon the re-incorporation of ubiquinone to the levels found in the lyophilized particles before extraction with pentane. Partial (about 50%) extraction of ubiquinone resulted in markedly decreased succinate and NADH oxidase activities.

3
Added cytochrome c did not replace ubiquinone in restoring the succinate or NADH oxidase activity of ubiquinone-depleted particles. It stimulated the NADH oxidase, but not the succinate oxidase, activity of the “ubiquinone-incorporated” particles, but the same stimulation occurred with the lyophilized particles before ubiquinone extraction. The normal, lyophilized, and “ubiquinone-incorporated” particles contained equal amounts of both total and enzymatically reducible cytochromes.

4
In the presence of KCN, NADH reduced the cytochromes, including cytochrome b, only at insignificant rates in the ubiquinone-depleted particles as compared to the normal and lyophilized preparations, and these rates were greatly stimulated upon the re-incorporation of ubiquinone. Succinate caused a rapid partial (about 25%) reduction of cytochrome b, but not of the rest of the cytochromes, in the ubiquinone-depleted particles. This reduction occurred also in the absence of KCN, and the fraction of cytochrome b, so reduced was not reoxidized when succinate oxidation was inhibited by malonate. Evidence for the occurrence of such an enzymatically non-oxidizable form of cytochrome b was also obtained in the normal, lyophilized and “ubiquinone-incorporated” particles, but, in those cases, this cytochrome b was reduced by both succinate and NADH. In the presence of antimycin A, all cytochrome b in the ubiquinone-depleted particles was rapidly reduced by succinate but not by NADH.

5
The normal and lyophilized particles catalyzed a rotenone-sensitive oxidation of NADH by fumarate. This reaction was completely absent from the ubiquinone-depleted particles and was restored upon the re-incorporation of ubiquinone.

6
N,N,N′,N′-Tetramethyl-p-phenylenediamine catalyzed an NADH and succinate oxidase activity in antimycin A-inhibited particles. This NADH oxidase activity was partially sensitive to rotenone in the normal, lyophilized and “ubiquinone-incorporated” particles, but completely rotenone-insensitive in the ubiquinone-depleted particles. All four types of particles were active in catalyzing the antimycin A-sensitive oxidation of menadiol.

7
It is concluded that uniquinone is essential for the interaction of succinate dehydrogenase, NADH dehydrogenase and cytochrome b, and that this interaction is a requisite for the normal function of the respiratory chain. Functionally modified forms of cytochrome b, arising as a consequence of structural damage or antimycin A treatment, are discussed in relation to existing information and proposals concerning the role of cytochrome b and ubiquinone in electron transport.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1969.tb00609.x

Studies with ubiquinone-depleted submitochondrial particles. Effects of extraction and reincorporation of ubiquinone on the kinetics of succinate dehydrogenase.

Studies with ubiquinone-depleted submitochondrial particles. Essentiality of ubiquinone for the interaction of succinate dehydrogenase, NADH dehydrogenase, and cytochrome b.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2869%2980086-4

In-Young Lee

Papers

Studies with ubiquinone-depleted submitochondrial particles