The metal-free hydrogenase from methanogenic archaea: evidence for a bound cofactor

The hmd gene, which encodes the metal-free hydrogenase in methanogenic archaea, was heterologously expressed in Escherichia coli. The overproduced enzyme was completely inactive. High activity could, however, be induced by the addition of ultrafiltrate from active enzyme denatured in 8 M urea. The active fraction in the ultrafiltrate was heat-labile and migrated on gel filtration columns with an apparent molecular mass well below 1000 Da.