Artificial Maturation of the Highly Active Heterodimeric [FeFe] Hydrogenase from Desulfovibrio desulfuricans ATCC 7757 |
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Authors: | James A Birrell Kathrin Wrede Krzysztof Pawlak Patricia Rodriguez-Maciá Olaf Rüdiger Edward J Reijerse Wolfgang Lubitz |
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Affiliation: | Max Planck Institute for Chemical Energy Conversion, Stiftstraße 34–36, D-45470 Mülheim an der Ruhr, Germany |
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Abstract: | Hydrogenases catalyze the reduction of protons and oxidation of molecular hydrogen with high turnover frequencies and low overpotentials under ambient conditions. The heterodimeric FeFe] hydrogenase from Desulfovibrio desulfuricans has an exceptionally high activity, and can be purified aerobically in an oxygen-stable inactive state. Recently, it was demonstrated that monomeric FeFe] hydrogenases produced recombinantly in Escherichia coli can be artificially maturated by simply incubating the inactive “apo” enzymes with the synthetic 2Fe] cofactor mimic Fe2(adt)(CO)4(CN)2]2−. Here, we use the same technique to produce the heterodimeric “apo” hydrogenase from D. desulfuricans in E. coli with a high yield and purity, and maturate the “apo” enzyme with Fe2(adt)(CO)4(CN)2]2− to generate fully active “holo” enzyme. Interestingly, the rate of the artificial maturation process with D. desulfuricans is significantly slower than that for all other hydrogenases tested so far. The artificially maturated enzyme is spectroscopically and electrochemically identical to the native enzyme and shows high rates of hydrogen production (3700 s−1) and hydrogen oxidation (63,000 s−1). We expect that our highly efficient production method will facilitate future studies of this enzyme and other related FeFe] hydrogenases from Desulfovibrio species. |
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Keywords: | biocatalysis EPR spectroscopy IR spectroscopy metalloenzymes protein expression |
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