Selenium-dependent clostridial glycine reductase.

The two membrane-associated protein components of the clostridial glycine reductase system were treated with detergents and separated by chromatography on DEAE-cellulose. Each was then further purified by chromatographic procedures. Protein B, obtained in highly purified form after loo-fold enrichment, contains a carbonyl group cofactor essential for its catalytic activity. The ability of this protein to catalyze a slow exchange of hydrogen of the methylene group of glycine with the solvent suggests the participation of a Schiff base intermediate formed by interaction of the amino group of glycine and a carbonyl group on protein B. This exchange reaction serves as a convenient independent assay of protein B when tritium-labeled glytine or 3Hz0 is employed. A second membrane-associated protein present in fraction C, that was enriched about 35-fold, appears to co-purify with iron. Recombination of protein B, fraction C, and homogeneous selenoprotein A reconstitutes the active glycine reductase complex. All three components are essential for the reduction of glycine by a dithiol to acetate and ammonia. The enzyme system, which catalyzes the concomitant esterification of orthophosphate, specifically requires ADP as acceptor when reconstituted from proteins freed of contaminating adenylate kinase. As established previously with impure enzyme fractions, 1 mol of ATP is synthesized from orthophosphate and ADP per mol of glycine reduced to 1 mol each of acetate and ammonia.