FAD requirement for the reduction of coenzyme F420 by hydrogenase from Methanobacterium formicicum

Hydrophobic interaction chromatography of coenzyme F420-reducing hydrogenase purified from Methanobacterium formicicum depleted protein-bound FAD and eliminated the ability to reduce coenzyme F420. Preincubation of the FAD-depleted hydrogenase with FAD restored 85% of the coenzyme F420-reducing activity. FMN did not replace FAD. A Kd of 12 microM was estimated for FAD. Analysis of the reactivated hydrogenase following molecular sieve column chromatography showed that FAD was bound to protein. The results indicate that protein-bound FAD is reversibly removed from the coenzyme F420-reducing hydrogenase and that this flavin is required for the reduction of coenzyme F420.