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Electric-Field-Induced Redox Potential Shifts of Tetraheme Cytochromes c₃ Immobilized on Self-Assembled Monolayers: Surface-Enhanced Resonance Raman Spectroscopy and Simulation Studies

Laura Rivas ^* , Cláudio M. Soares ^*, António M. Baptista ^*, Jalila Simaan ^*, Roberto E. Di Paolo ^*, Daniel H. Murgida ^*  and Peter Hildebrandt ^* 

^* Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal; and Technische Universität Berlin, Institut für Chemie, D-10623 Berlin, Germany

Correspondence: Address reprint requests to Peter Hildebrandt, Tel.: 493031421419; Fax: 493031421122; E-mail: hildebrandt{at}chem.tu-berlin.de; or Cláudio M. Soares, Tel.: 351214469610; Fax: 351214411277; E-mail: claudio{at}itqb.unl.pt.

The tetraheme protein cytochrome c₃ (Cyt-c₃) from Desulfovibrio gigas, immobilized on a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid, is studied by theoretical and spectroscopic methods. Molecular dynamics simulations indicate that the protein docks to the negatively charged SAM via its lysine-rich domain around the exposed heme IV. Complex formation is associated with only little protein structural perturbations. This finding is in line with the resonance Raman and surface-enhanced resonance Raman (SERR) spectroscopic results that indicate essentially the same heme pocket structures for the protein in solution and adsorbed on SAM-coated Ag electrodes. Electron- and proton-binding equilibrium calculations reveal substantial negative shifts of the redox potentials compared to the protein in solution. The magnitude of these shifts decreases in the order heme IV (–161 mV) > heme III (–73 mV) > heme II (–57 mV) > heme I (–26 mV), resulting in a change of the order of reduction. These shifts originate from the distance-dependent electrostatic interactions between the SAM headgroups and the individual hemes, leading to a stabilization of the oxidized forms. The results of the potential-dependent SERR spectroscopic analyses are consistent with the theoretical predictions and afford redox potential shifts of –160 mV (heme IV), –90 mV (heme III), –70 mV (heme II), and +20 mV (heme I) relative to the experimental redox potentials for Cyt-c₃ in solution. SERR spectroscopic experiments reveal electric-field-induced changes of the redox potentials also for the structurally very similar Cyt-c₃ from Desulfovibrio vulgaris, although the shifts are somewhat smaller compared to Cyt-c₃ from D. gigas. This study suggests that electric-field-induced redox potential shifts may also occur upon binding to biomembranes or partner proteins and thus may affect biological electron transfer processes.