Oxidative stress is considered a major contributor to the etiology of both "normal" senescence and severe pathologies with serious public health implications. Several cellular sources, including mitochondria, are known to produce significant amounts of reactive oxygen species (ROS) that may contribute to intracellular oxidative stress. Mitochondria possess at least 10 known sites that are capable of generating ROS, but they also feature a sophisticated multilayered ROS defense system that is much less studied. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal at the level of mitochondria. An integrative systemic approach is applied to analysis of mitochondrial ROS metabolism, which is "dissected" into ROS generation, ROS emission, and ROS scavenging. The in vitro ROS-producing capacity of several mitochondrial sites is compared in the metabolic context and the role of mitochondria in ROS-dependent intracellular signaling is discussed.

The Role of Mitochondria in Reactive Oxygen Species Metabolism and Signaling

http://www.jbc.org/content/291/27/14274.full.pdf

Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to Modulate Intracellular Succinate Levels.

Publisher Summary This chapter discusses the four aspects of mammalian succinate dehydrogenase: (1) a critical comparison of assay methods, (2) activation–deactivation of the enzyme, (3) the active site of the enzyme, and (4) the comparison of the properties of various purified preparations including recent improvements of procedures for isolating the reconstitutively active form in high yield and with a high turnover number. Because the catalytic turnover of succinate dehydrogenase is faster than the rate-limiting step in the respiratory chain, artificial electron acceptors are usually used for assays of the enzyme in order to ensure that full activity is being measured. Of these, phenazine methosulfate (PMS) with either DCIP or cytochrome c as the terminal oxidant, may be used with the particulate or soluble preparations. Ferricyanide has been widely used for the assay of succinate dehydrogenase. Polarographic or manometric measurements of the succinate–PMS reaction are not recommended, as they are less sensitive and the rate is limited by the oxygen concentration.

Mammalian succinate dehydrogenase.

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.

Publisher Summary This chapter discusses the control of oxidative metabolism by adenosine diphosphate (ADP) availability in isolated mitochondria and perfused organs. It focuses on the control of dehydrogenase activity. The enzymes, such as, the pyruvate dehydrogenase complex and some of the enzymes of the tricarboxylate cycle have been considered. The most important characteristics of these enzymes are that (1) they catalyze non-equilibrium reactions; (2) exert a dominant influence over the flux through the pathways that are central to catabolic metabolism; and (3) they are mitochondrial. This localization means that the supply of substrate, and dissipation of product, might be limited by the permeability properties of the inner mitochondrial membrane. Because these enzymes are dehydrogenases, the final arbiter of their activity is the respiratory chain, with its coupling to the phosphorylation of ADP. The chapter describes the control of the tricarboxylate cycle and of pyruvate dehydrogenase in their mitochondrial context

L. Wojtczak

Papers

The inhibition of succinate dehydrogenase by oxaloacetate