Improved purification, crystallization and crystallographic study of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp S-77

The purification procedure of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77 was improved by applying treatment with trypsin before chromatography. Purified protein samples both with and without trypsin treatment were successfully crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol as a precipitant. Both crystals belonged to space group P2(1), with unit-cell parameters a = 63.90, b = 118.89, c = 96.70 angstrom, beta = 100.61 degrees for the protein subjected to trypsin treatment and a = 65.38, b = 121.45, c = 98.63 angstrom, beta = 102.29 degrees for the sample not treated with trypsin. The crystal obtained from the trypsin-treated protein diffracted to 1.60 angstrom resolution, which is considerably better than the 2.00 angstrom resolution obtained without trypsin treatment. The [NiFe]-hydrogenase from Citrobacter sp. S-77 retained catalytic activity with some amount of O-2, indicating that it has clear O-2 tolerance.