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          Institute: MPI für bioanorganische Chemie     Collection: MPI für bioanorganische Chemie     Display Documents



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ID: 713325.0, MPI für bioanorganische Chemie / MPI für bioanorganische Chemie
Hybrid [FeFe]-Hydrogenases with Modified Active Sites Show Remarkable Residual Enzymatic Activity
Authors:Siebel, Judith F.; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward J.; Lubitz, Wolfgang
Language:English
Date of Publication (YYYY-MM-DD):2015
Title of Journal:Biochemistry
Journal Abbrev.:Biochem.
Volume:54
Issue / Number:7
Start Page:1474
End Page:1483
Review Status:Internal review
Audience:Experts Only
Abstract / Description:[FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe-2(adt)(CO)(3)(CN)(2) (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN- ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed similar to 50% of the native enzyme activity. This would suggest that the CN- ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Bronsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme.
Comment of the Author/Creator:Date: 2015, FEB 24 2015
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für bioanorganische Chemie
Identifiers:ISI:000350193800005 [ID No:1]
ISSN:0006-2960 [ID No:2]
DOI:10.1021/bi501391d [ID No:3]
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