18 1. MALONATE 



flavin and nonheme iron are reduced by succinate, and the functional role 

 of iron in the catalysis is further indicated by the inhibition produced by 

 complexing the iron with 1,10-phenanthroline or /J^-globulin (Singer et al., 

 1957). Electron spin resonance studies of succinate oxidase have demonstrat- 

 ed signals when succinate is added; these free radicals seem to be associat- 

 ed with the dehydrogenase and possibly reflect changes in the states of 

 iron or flavin (Commoner and Hollocher, 1960; Hollocher and Commoner, 

 1960). The high sensitivity of succinate dehydrogenase to most sulfhydryl 

 reagents indicates the presence of an SH group at or near the succinate- 

 binding site. One may, therefore, characterize succinate dehydrogenase 

 from our present knowledge as containing two cationic groups for the 

 binding of succinate, an SH group nearby, some nonheme iron, and a unique 

 flavin dinucleotide in a tight peptide complex. 



Relationship of Malonate Inhibition to the Electron Acceptor Used 



Measurement of malonate inhibition involves either the oxygen uptake of 

 the complete succinate oxidase system, or the determination spectroscopi- 

 cally of the reduction of one of the normal components (such as cytochrome 

 c), or the reduction of some artificial electron-acceptor dye. The complete 

 system can reduce a variety of substances in the presence of succinate, and 

 malonate has been shown to inhibit such reductions whatever the acceptor 

 used: methylene blue (Quastel and Whetham, 1925; Hopkins et al., 1938; 

 Forssman, 1941; Franke 1944 a; Kaltenbach and Kalnitsky, 1951 a; Wad- 

 kins and Mills, 1955), ferricyanide (Stoppani, 1948; Thorn, 1953), tetrazolium 

 dyes (Barker, 1953; Becker and Rauschke, 1951; Zollner and Rothemund 

 1954; Waterhouse, 1955), manganese dioxide (Hochster and Quastel, 1952), 

 2,6-dichlorophenolindophenol (Repaske, 1954; Wadkins and Mills, 1955, 

 Millerd, 1951), janus red (Agosin and von Brand, 1955), brilliant cresyl 

 blue (Agosin and von Brand, 1955), and lY-methylphenazine sulfate (Singer 

 et al., 1956 b). Inhibition of cytochrome c reduction has also been observed 

 Seaman, 1954). There is thus substantial evidence that malonate blocks 

 electron flow very early in the sequence, as would be expected if it prevents 

 the binding of succinate to the dehydrogenase. The most proximal location 

 of the site of malonate inhibition comes from the work of Ziegler (1961) on 

 electron transport particles from heart mitochondria. Some of the nonheme 

 iron is reduced by succinate and this is blocked by 20 raM malonate. It 

 may also be noted that succinate reduces NAD and NADP in submito- 

 chondrial particles from heart through an ATP-dependent system and this 

 is readily inhibited by malonate (Snoswell, 1962; Hommes, 1963; Lee et al., 

 1964), which possibly indicates that succinate dehydrogenase is involved. 

 The addition of malonate to succinate dehydrogenase brings about changes 

 in the absorption spectrum in the flavin region, as do succinate, fumarate, 

 and other competitive inhibitors (Dervartanian and Veeger, 1962). There is 



