Crystallization and preliminary X-ray analysis of the NAD(+)-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1

摘要

NAD(+)-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD(+) and NADH. Here, the isolation, purification and crystallization of the NAD(+)-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD(+)-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 angstrom resolution and belonged to space group C2, with unit-cell parameters a = 131.43, b = 189.71, c = 124.59 angstrom, = 109.42 degrees. Assuming the presence of two NAD(+)-reducing [NiFe] hydrogenase molecules in the asymmetric unit, V-M was calculated to be 2.2 angstrom(3)Da(-1), which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.