Properties of formate dehydrogenase in Methanobacterium formicicum

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Abstract
Soluble formate dehydrogenase from M. 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 .mu.mol of methyl viologen/min per mg of protein and 8.2 .mu.mol of coenzyme F420/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 .mu.M) than for coenzyme F420 (6 .mu.M). The purified enzyme also reduced FMNH2 (Km = 13 .mu.M) and FAD (Km = 25 .mu.M) 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.degree. C. The MW was 288,000, as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 .mu.M), azide (Ki = 39 .mu.M), .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.

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