Chemical assembly of multiple metal cofactors: The heterologously expressed multidomain [FeFe]-hydrogenase from Megasphaera elsdenii

• A purely chemical methodology for assembly of hydrogenase metal cofactors and recombinant enzyme activation is reported.
• The first complete EPR and FTIR characterization of the hydrogenase from Megasphaera elsdenii
• EPR and FTIR spectroscopy allowed for the identification of Hox, Hox-CO and Hred states for the hydrogenase from M. elsdenii

[FeFe]-hydrogenases are unique and fascinating enzymes catalyzing the reversible reduction of protons into hydrogen. These metalloenzymes display extremely large catalytic reaction rates at very low overpotential values and are, therefore, studied as potential catalysts for bioelectrodes of electrolyzers and fuel cells. Since they contain multiple metal cofactors whose biosynthesis depends on complex protein machineries, their preparation is difficult. As a consequence still few have been purified to homogeneity allowing spectroscopic and structural characterization. As part of a program aiming at getting easy access to new hydrogenases we report here a methodology based on a purely chemical assembly of their metal cofactors. This methodology is applied to the preparation and characterization of the hydrogenase from the fermentative anaerobic rumen bacterium Megasphaera elsdenii, which has only been incompletely characterized in the past.

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