Beef heart succinate dehydrogenase

Massey, V. Studies on succinic dehydrogenase. VII. Valency state of the iron in beef heart succinic dehydrogenase. J. Biol. Chem. 229, 763—770 (1957). 

Trinuclear clusters have been detected in over 20 proteins as well as a number of enzymes, among them aconitase, beef heart succinate-ubiquinone oxidoreductase (120), Escherichia coli nitrate reductase (121), E. coli fumarate reductase (122), and succinate dehydrogenase (123). Selected instances of the occurrence of [3Fe-4S] clusters are listed in Table II. Because of the paramagnetic ground states of both oxidation levels, these clusters can be uniquely identified by a number of spectroscopic techniques. Among these, Mossbauer spectroscopy in applied magnetic fields (124, 128, 132, 141-143) and low temperature MCD spectroscopy (127, 138, 144-146) are decisive. While there are small spectroscopic differences among certain [3Fe-4S] centers, the similarities dominate and support the essential structure 3 for all. In a number of the earlier papers on protein... 

At the active site of beef heart mitochondrial succinate dehydrogenase (EC 1.3.99.1), the FAD is covalently linked by C-8a to nitrogen of a histidine residue (Fig. 2b) [20]. In the catalytic reaction, removal of a proton from C-3 of the succinate may be followed by attack of the 3-carbanion on N-5 of the FAD to form an intermediate adduct, which breaks down with loss of a proton from C-2 of the succinate, giving fumarate and a reduced FAD moiety [21]. This mechanism is not certain, but it is established that the succinate loses two non-equivalent hydrogen atoms by a trans elimination (Fig. 4) [22], In other enzymes, different types of covalent attachment of the FAD are known [23]. 

The rate with the quinone is less than 1% of the rate with TMPD so that it would be interesting to determine whether the in-vivo rate of quinone turnover during succinate oxidation is consistent with the operation of the electron transport system. Although the Sulfolobus enzyme is composed of four subunits, there is no evidence that all are associated with the enzyme other than that they copurify with enzyme activity. The succinate dehydrogenase from eucarya and bacteria have two subunits whose M, are approximately 70000 and 28 000 [112]. They appear to be analogous to the M, 66000 and 31 000 subunits from the Sulfolobus succinic dehydrogenase. The M, 66 000 subunit from Sulfolobus is catalytically active by itself, and like the Mr 70000 subunit contains covalently-bound FAD. Antisera against the Mr 66000 Sulfolobus subunit cross-reacts with an Mr 67 000 constituent in membranes from T. acidophilum, S. solfataricus, beef-heart submitochondrial particles, and Bacillus subtilis. 

Succinate dehydrogenase is structurally and functionally very closely related to the fumarate reductase from E. coli, indeed the catalytic subcomplex SdhAB lacking the membrane anchors can be obtained from beef heart mitochondria and other sources. SdhAB can be adsorbed at a PGE electrode, although it has proven difficult to obtain films that have a sufficiently high coverage to observe non-tumover signals. Even so, some interesting catalytic activity is revealed, as shown in Fig. 4-8. 

The clear detection of both reversible active-site and biocatalytic waves represents a completeness that establishes the feasibility of applying direct elec-tochemistry to probe the mechanism of action of complex redox enzymes. To take this further, I shall take up the author s prerogative for mentioning studies currently underway in the laboratory and mention, briefly, another membrane-bound enzyme, fumarate reductase (FR), isolated from Escherichia coll Structurally, this is closely related to the more familiar succinate dehydrogenase (SDH) which constitutes the major part of Complex II of the mitochondrial respiratory chain. Of the four subunits which make up the membrane-bound system, two may be freed to give a soluble enzyme that is active in fumarate reduction by artificial electron donors [230]. The larger of these, MW approx. 70000, contains, like SDH, a covalently bound FAD. The smaller, MW approx. 30000, appears to contain three Fe-S clusters. These are termed centre 1 ([2Fe-2S]), centre 2 ([4Fe-4S]) and centre 3 ([3Fe-4S]). Their respective reduction potentials as determined by potentiometry are — 20 mV, — 320 mV, and — 70 mV [231], (Although the potential of the FAD has not been determined for FR, the two-electron value for beef heart SDH is — 79 mV at pH 7.0. The radical form is unstable since the two one-electron reductions occur at potentials of — 127 and — 31 mV respectively [232].)... 

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