title: Role of the HoxZ subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha immobilized on electrodes.
abstract: The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode.
authors.affiliation: Insitut für Chemie, Technische Universität Berlin, Strasse des 17, Juni 135, 10623 Berlin, Germany.
authors.forename: Murat
authors.initials: M
authors.lastname: Sezer
authors.forename: Stefan
authors.initials: S
authors.lastname: Frielingsdorf
authors.forename: Diego
authors.initials: D
authors.lastname: Millo
authors.forename: Nina
authors.initials: N
authors.lastname: Heidary
authors.forename: Tillman
authors.initials: T
authors.lastname: Utesch
authors.forename: Maria-Andrea
authors.initials: MA
authors.lastname: Mroginski
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.forename: Inez M
authors.initials: IM
authors.lastname: Weidinger
mesh_terms: D055162:Biocatalysis; D002462:Cell Membrane / Q000378:metabolism*; D020118:Cupriavidus necator / Q000201:enzymology*; D045303:Cytochromes b / Q000737:chemistry / Q000378:metabolism*; D004566:Electrodes; D004579:Electron Transport; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D013059:Spectrum Analysis, Raman; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D021122:Protein Subunits; D045303:Cytochromes b; C043296:nickel-iron hydrogenase; D006864:Hydrogenase