Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b.

A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase.