The nature of the cofactor requirements of the hydrogenase system from Clostridium kluyveri.

Previous studies in this laboratory showed that the reduction of diphosphopyridine nucleotide with molecular hydrogen, as catalyzed by extracts of C~ostridium kluyveri, involves the participation of at least two protein components (1, Z), one of which is ferredoxin (3), and of at least two cofactors obtainable from boiled cell extracts (4). The heat-stable cofactors were separated by column chromatography on Magnesol-Celite (4) ; one was identified as flavin adenine dinucleotide; the other, referred to as Magnesol cofactor,i has not been identified. This report describes further resolution of the Magnesol cofactor preparation into two subfractions that are independently capable of activating the enzyme system. The active principles in these fractions could not be identified with any of the common nucleotide derivatives, but they may be replaced by relatively high concentrations of flavin mononucleotide, adenosine triphosphate, or any of a large number of dior trinucleotide derivatives. Evidence is presented indicating that these substances serve as activators of the hydrogenase system. A preliminary report of this work has been published (5).