title: Lyophilization protects [FeFe]-hydrogenases against O2-induced H-cluster degradation.
abstract: Nature has developed an impressive repertoire of metal-based enzymes that perform complex chemical reactions under moderate conditions. Catalysts that produce molecular hydrogen (H2) are particularly promising for renewable energy applications. Unfortunately, natural and chemical H2-catalysts are often irreversibly degraded by molecular oxygen (O2). Here we present a straightforward procedure based on freeze-drying (lyophilization), that turns [FeFe]-hydrogenases, which are excellent H2-producers, but typically extremely O2-sensitive in solution, into enzymes that are fully resistant against O2. Complete dryness protects and conserves both, the [FeFe]-hydrogenase proteins and their inorganic active-site cofactor (H-cluster), when exposed to 100% O2 for days. The full H2-formation capacity is restored after solvation of the lyophilized enzymes. However, even minimal moisturizing re-establishes O2-sensitivity. The dry [FeFe]-hydrogenase material is superior also for advanced spectroscopic investigations on the H-cluster reaction mechanism. Our method provides a convenient way for long-term storage and impacts on potential biotechnological hydrogen production applications of hydrogenase enzymes.
authors.affiliation: Ruhr-Universität Bochum, Fakultät für Biologie und Biotechnologie, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, 44801 Bochum, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Noth
authors.affiliation: Freie Universität Berlin, Institut für Experimentalphysik, 14195 Berlin, Germany.
authors.forename: Ramona
authors.initials: R
authors.lastname: Kositzki
authors.affiliation: Ruhr-Universität Bochum, Fakultät für Chemie und Biochemie, Anorganische Chemie I-Bioanorganische Chemie, 44801 Bochum, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Klein
authors.affiliation: Ruhr-Universität Bochum, Fakultät für Biologie und Biotechnologie, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, 44801 Bochum, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Winkler
authors.affiliation: Freie Universität Berlin, Institut für Experimentalphysik, 14195 Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Haumann
authors.affiliation: Ruhr-Universität Bochum, Fakultät für Biologie und Biotechnologie, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, 44801 Bochum, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Happe
mesh_terms: D020134:Catalytic Domain; D003013:Clostridium / Q000201:enzymology; D005612:Freeze Drying; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012996:Solutions / Q000737:chemistry; D056928:X-Ray Absorption Spectroscopy
chemical_list: D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D012996:Solutions; D006859:Hydrogen; C118830:iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen