The preparation and properties of a soluble diphosphopyridine nucleotide cytochrome c reductase.

The transfer of electrons from the pyridine nucleotide-linked dehydrogenases to cytochrome c is mediated by a class of enzymes which has been termed the cytochrome c reductases (1). Two TPNH cytochrome c reductases, both flavoproteins, have been purified, one by Haas et al. (1) from yeast, and another by Horecker (2) from liver.’ Analogous enzymes in the case of DPNH have been implicated in particulate preparations (3) and crude extracts (4, 5), but our understanding of their nature and function is still fragmentary. The relationship between the pyridine nucleotide-cytochrome c reductases and the diaphorase discovered by von Euler et al. (6,7) and Green and Dewan (8), and isolated in purified form by Straub (9), remains obscure. This enzyme catalyzes the reoxidation of DPKH by dyes, such as methylene blue, but is incapable of interaction with cytochrome c, the physiological oxidant. This inability may be due to a structural modification of a native cytochrome c reductase (3, 10). On the basis of inhibiton studies, Slater (11) has concluded that a factor sensitive to BAL (2,3-dimercaptopropanol) is necessary for full reductase activity. More recently Potter and Reif (12) have implicated a similar factor, as a result of their studies on antimycin A inhibition. From this laboratory Hayaishi (13) has already reported on the preparation and assay of a soluble DPN cytochrome c reductase from pigeon breast muscle. This paper deals with an improved method for the preparation of this reductase.