Properties of formate dehydrogenase in Methanobacterium formicicum.
Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D019605:Euryarchaeota / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010100:Oxygen / Q000494:pharmacology; D012256:Riboflavin / Q000031:analogs & derivatives / Q000378:metabolism; D013696:Temperature
D005182:Flavin-Adenine Dinucleotide; C015084:coenzyme F420; D005486:Flavin Mononucleotide; D005560:Formate Dehydrogenases; D000445:Aldehyde Oxidoreductases; D010100:Oxygen; D012256:Riboflavin