Purification and partial characterisation of a reversible artificial mediator accepting NADH oxidoreductase from Clostridium thermoaceticum

An NAD(H)-dependent artificial mediator accepting pyridine nucleotide oxidoreductase present in Clostridium thermoaceticum has been purified 50-fold by three chromatographic steps to apparent electrophoretical homogeneity with a yield of 25%. By PAGE and gel filtration the molecular mass of the native enzyme was estimated to be 300 kDa and 210 kDa, respectively. By SDS/gel electrophoresis, a single band was found at 17 000 Da, suggesting a homododecamer. Reducing carbamoylmethylviologen or hexa-cyanoferrate(III) with NADH, the enzyme was most active at pH 10 and the specific activities were 100 mu mol min(-1) mg(-1) protein and 800 mu mol min(-1) mg(-1) protein, respectively. The K-m values for hexacyanoferate(III), carbamoylmethylviologen and NADH at pH 8.5 were determined to be 0.40, 0.55 and 1.1 m.M, respectively, Other electron accepters for the dehydrogenation of NADH were 2,6-dichlorophenolindophenol anthraquinone-2,6-disulphonate, ubiquinone 0 and FAD. In the reduction of NAD(+) with reduced methyl viologen (MV(+.)), the specific activity was about 225 mu mol min(-1) mg(-1) protein at the pH maximum of 5.0. The K-m values fur reduced methylviologen, NADH and NAD(+) were 1.0, 1.1 and 0.25 mM, respectively. The enzyme had 10.6 atoms iron and 12.7 atoms sulphur per dodecamer. A significant content of flavin or molybdopterin cofactor could not be detected. The first 45 amino acids of the oxidoreductase show a surprisingly high degree of identity or similarity with the ribosomal L12 protein of various eubacteria, the acyl carrier proteins of microorganisms. but also with bovine heart mitochondria and a 3-phosphoglycerate dehydrogenase as well as a gyceraldehyde 3-phosphate dehydrogenase from bacteria and pea chloroplasts, respectively.