Abstract
Abstract The DPNH dehydrogenase of the mitochondrial electron transport system has been obtained in a highly purified, soluble form after resolution of DPNH-coenzyme Q reductase (Complex I) particles by chaotropic agents. The enzyme contains 1 mole of flavin mononucleotide, 4 g atoms of iron, and 4 moles of acid-labile sulfide per 70,000 g. The soluble, but not the particle-bound, enzyme behaves as a diaphorase and catalyzes the unspecific reduction of quinones and ferric compounds such as menadione, 2,6-dichloroindophenol, coenzymes Q, ferricyanide, and cytochrome c. Mercurials inhibit all the reductase activities of the soluble dehydrogenase, and DPNH (g10-4 m) inhibits its menadione and coenzyme Q reductase activities. The structure and function of DPNH dehydrogenase in both the soluble and the particle-bound form are discussed.
Highlights
The DPNH dehydrogenase of the mitochondrial electron transport system has been obtained in a highly purified, soluble form after resolution of DPNH-coenzyme
General Considerations-Previous work from this laboratory has shown that the mitochondrial electron transport system can be divided into its component enzyme complexes by application of a general procedure involving low levels of bile salts, KCl, ammonium acetate, and ammonium sulfate [9]
High concentrations of bile salts at elevated temperatures can bring about the resolution of Complex III into a cytochrome b-rich and a cytochrome cl-rich fraction [19], or heat-acid-ethanol treatment can dissociate the DPNH dehydrogenase of Complex I from mitochondrial particles
Summary
The DPNH dehydrogenase of the mitochondrial electron transport system has been obtained in a highly purified, soluble form after resolution of DPNH-coenzyme. This category includes the dehydrogenase preparations which have a molecular weight between 70,000 and 140,000, contain flavin iron and acid-labile sulfide in a ratio of 2 to 4 iron atoms and 2 to 4 labile sulfide groups per flavin moiety, and can react with ferric complexes (ferricyanide and cytochrome c) and quinoid compounds (menadione, 2, &dichloroindophenol, and coenzyme Q) as electron acceptors With one exception, these dehydrogenases [14] have been extracted from tissue homogenates or mitochondrial particles in the presence of 9 to 11 y0 ethanol at pH 4.8 to 5.3 and 4345”, a procedure originally devised for isolation of the Straub diaphorase [7]. These changes are consistent with the possibility of a conformation of the active site more accessible to DPNH and electron acceptors
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