| Autor: |
Hartmann T; Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam , Karl-Liebknecht Strasse 24-25, 14476 Potsdam, Germany., Schrapers P; Institute of Experimental Physics, Freie Universität Berlin , Arnimallee 14, 14195 Berlin, Germany., Utesch T; Institute of Chemistry, Technical University of Berlin , Straße des 17. Juni135, 10623 Berlin, Germany., Nimtz M; Helmholtz Center for Infection Research , Inhoffenstraße 7, 38124 Braunschweig, Germany., Rippers Y; Institute of Chemistry, Technical University of Berlin , Straße des 17. Juni135, 10623 Berlin, Germany., Dau H; Institute of Experimental Physics, Freie Universität Berlin , Arnimallee 14, 14195 Berlin, Germany., Mroginski MA; Institute of Chemistry, Technical University of Berlin , Straße des 17. Juni135, 10623 Berlin, Germany., Haumann M; Institute of Experimental Physics, Freie Universität Berlin , Arnimallee 14, 14195 Berlin, Germany., Leimkühler S; Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam , Karl-Liebknecht Strasse 24-25, 14476 Potsdam, Germany. |
| Abstrakt: |
Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase. |
