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Spectroelectrochemical Study of the [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F in Solution and Immobilized on Biocompatible Gold Surfaces
Zitatschlüssel ISI:000271580700030
Autor Millo, Diego and Pandelia, Maria-Eirini and Utesch, Tillmann and Wisitruangsakul, Nattawadee and Mroginski, Maria A. and Lubitz, Wolfgang and Hildebrandt, Peter and Zebger, Ingo
Seiten 15344-15351
Jahr 2009
ISSN 1520-6106
DOI 10.1021/jp906575r
Adresse 1155 16TH ST, NW, WASHINGTON, DC 20036 USA
Jahrgang 113
Nummer 46
Monat NOV 19
Zusammenfassung The catalytic cycle of the anaerobic [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) both in solution and immobilized on an Au electrode was studied by IR spectroscopic and electrochemical methods. IR spectroelectrochemistry in solution at different pH values allows the identification of the various redox-states of the active site and the determination of the midpoint potentials, as well as their acid-base equilibria. The spectroscopic characterization was based on the unique marker bands of the CN and CO stretchin a modes of the Ni-Fe center and served as reference for the surface-enhanced IR absorption (SEIRA) study of the immobilized enzyme. Using structural models of hydrogenases from DvMF and Desulfovibrio gigas, dipole moment calculations were carried out to guide the immobilization strategy. In view of the high dipole moment of about 1100 D pointing through the negatively charged area surrounding the distal [FeS] cluster, the Au electrode was coated by a self-assembled monolayer of amino-terminated mercaptanes which, due to the positively charged head groups, permit a durable electrostatic binding of the protein, SEIRA spectroscopy revealed a structurally and functionally intact active site as demonstrated by the reversible activation and inactivation under hydrogen and argon, respectively. Cyclic voltammetry on the immobilized enzyme demonstrate a reversible anaerobic inactivation upon changing the applied potential. The ``switch'' potential (E-switch) associated with the reductive reactivation was determined to be -33 mV (vs normal hydrogen electrode). However, the catalytic current decreased on the time scale of hours during continuous cycling. SEIRA experiments demonstrate that the loss of catalytic activity is not due to protein desorption but is rather related to a slow degradation of the active site, possibly initiated by the attack of reactive species electrochemically generated from residual traces of oxygen in solution.
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