Studies on the Mechanism of Microsomal Triphosphopyridine Nucleotide-Cytochrome

Flavoproteins have long been recognized as hydrogen and electron carriers between pyridine nucleotides and cytochromes. Cytochrome reductases the oxidation-reduction mechanisms of which have been extensively studied (1) have contained metals, as have other flavin enzymes with cytochrome reductase activity, such as xanthine oxidase (a), liver aldehyde oxidase (3), and Neurospora (4) and Pseudomonas (5) nitrate reductases. However, information remains meager concerning the mechanism of metal-free flavin catalysis of electron transport between 2electron donors such as pyridine nucleotides and l-electron acceptors such as cytochromes. Microsomal reduced triphosphopyridine nucleotide-cytochrome c reductase is metal-free and may thus provide a suitable vehicle for studies of this type of catalysis. Williams and Kamin have reported the purification and some of the properties of pork liver microsomal TPNH-cytochrome c reductase (6). The enzyme was isolated both as a partially purified particulate preparation, by treatment of microsomes with aqueous butanol at pH 9, and as a soluble protein prepared by lipase treatment of microsomes and subsequent fractionation of the solubilized material. The enzyme was finally obtained as a preparation essentially homogeneous in the ultracentrifuge except for a small quantity (less than 10%) of a heavy componcnt. It was shown to contain flavin adenine dinucleotide as the prosthetic group, and the apoenzyme could be reactivated by either FAD or flavin mononucleotide. This as well as other data suggested that this enzyme was probably identical with Horecker’s (7) TPNH-cytochrome c reductase, isolated from