Continuous monitoring of the activation and activity of [NiFe]-hydrogenases by membrane-inlet mass spectrometry

The hydrogen-deuterium (H+/D-2) exchange reaction catalyzed by [NiFe]-hydrogenases in the D-2/H2O system has been used to study enzyme activation and activity by membrane-inlet mass spectrometry. The activation of the [NiFe]-hydrogenases from Thiocapsa roseopersicina (HynSL), Desulfovibrio fructosovorans (HynSL), Desulfmicrobium baculatum (HysSL), Rhodobacter capsulatus (HupUV), and of the bidirectional tetrameric HoxFUYH enzymes from Synechocystis PCC 6308 (Gloeocapsa alpicola) and Anabaena variabilis ATCC 29413 was determined in response to oxygen depletion and to reductant addition (molecular hydrogen, reduced methyl viologen). Natural physiological activators (NADH, NADPH) of the bidirectional [NiFe] hydrogenases could also be identified by the H+/D-2 exchange reaction. The data are discussed in the light of current models of hydrogenase catalytic mechanism. (C) 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.