title: [FeFe]-Hydrogenase with Chalcogenide Substitutions at the H-Cluster Maintains Full H2 Evolution Activity.
abstract: The [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii is particularly amenable to biochemical and biophysical characterization because the H-cluster in the active site is the only inorganic cofactor present. Herein, we present the complete chemical incorporation of the H-cluster into the HYDA1-apoprotein scaffold and, furthermore, the successful replacement of sulfur in the native [4FeH ] cluster with selenium. The crystal structure of the reconstituted pre-mature HYDA1[4Fe4Se]H protein was determined, and a catalytically intact artificial H-cluster variant was generated upon in vitro maturation. Full hydrogen evolution activity as well as native-like composition and behavior of the redesigned enzyme were verified through kinetic assays, FTIR spectroscopy, and X-ray structure analysis. These findings reveal that even a bioinorganic active site with exceptional complexity can exhibit a surprising level of compositional plasticity.
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Universitätsstrasse 150, 44801, Bochum, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Noth
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Universitätsstrasse 150, 44801, Bochum, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Esselborn
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biophysik, AG Spektroskopie, Universitätsstrasse 150, 44801, Bochum, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Güldenhaupt
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Universitätsstrasse 150, 44801, Bochum, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Brünje
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Universitätsstrasse 150, 44801, Bochum, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Sawyer
authors.affiliation: Ruhr Universität Bochum, Fakultät für Chemie und Biochemie, Lehrstuhl für Anorganische Chemie I/ Bioanorganische Chemie, Universitätsstrasse 150, 44801, Bochum, Germany. ulf.apfel@ruhr-uni-bochum.de.
authors.forename: Ulf-Peter
authors.initials: UP
authors.lastname: Apfel
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biophysik, AG Spektroskopie, Universitätsstrasse 150, 44801, Bochum, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biophysik, AG Röntgenstrukturanalyse an Proteinen, Universitätsstrasse 150, 44801, Bochum, Germany.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Hofmann
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Universitätsstrasse 150, 44801, Bochum, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Winkler
authors.affiliation: Ruhr Universität Bochum, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Universitätsstrasse 150, 44801, Bochum, Germany. thomas.happe@ruhr-uni-bochum.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Happe
keywords: H-clusters; H2 production; chalcogenide exchange; hydrogenases; selenium