Purification of the F420‐reducing hydrogenase from Methanosarcina barkeri (strain Fusaro)

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Abstract
The 8‐hydroxy‐5‐deazaflavin (coenzyme F420)‐reducing and methyl‐viologen‐reducing hydrogenase of the anaerobic methanogenic archaebacterium Methanosarcina barkeri strain Fusaro has been purified 64‐fold to apparent electrophoretic homogeneity. The purified enzyme had a final specific activity of 11.5 μmol coenzyme F420 reduced · min−1· mg protein−1 and the yield was 4.8% of the initial deazaflavin‐reducing activity. The hydrogenase exists in two forms with molecular masses of approximately 845 kDa and 198 kDa. Both forms reduce coenzyme F420 and methyl viologen and are apparently composed of the same three subunits with molecular masses of 48 kDa (α), 33 kDa (β) and 30 kDa (γ). The aerobically purified enzyme was catalytically inactive. Conditions for anaerobic reductive activation in the presence of hydrogen, 2‐mercaptoethanol and KCl or methyl viologen were found to yield maximal hydrogenase activity. Determination of the apparent Km of coenzyme F420 and methyl viologen gave values of 25 μM and 3.3 mM, respectively. The respective turnover numbers of the high molecular mass form of the hydrogenase are 353 s−1 and 9226 s−1.

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