Disentangling Electron Tunneling and Protein Dynamics of Cytochrome c through a Rationally Designed Surface Mutation

Abstract

Nonexponential distance dependence of the apparent electron-transfer (ET) rate has been reported for a variety of redox proteins immobilized on biocompatible electrodes, thus posing a physicochemical challenge of possible physiological relevance. We have recently proposed that this behavior may arise not only from the structural and dynamical complexity of the redox proteins but also from their interplay with strong electric fields present in the experimental setups and in vivo (J. Am Chem. Soc. 2010, 132, 5769-5778). Therefore, protein dynamics are finely controlled by the energetics of both specific contacts and the interaction between the protein's dipole moment and the interfacial electric fields. In turn, protein dynamics may govern electron-transfer kinetics through reorientation from low to high donor-acceptor electronic coupling orientations. Here we present a combined computational and experimental study of WT cytochrome c and the surface mutant K87C adsorbed on electrodes coated with self-assembled monolayers (SAMs) of varying thickness (i.e., variable strength of the interfacial electric field). Replacement of the positively charged K87 by a neutral amino acid allowed us to disentangle protein dynamics and electron tunneling from the reaction kinetics and to rationalize the anomalous distance dependence in terms of (at least) two populations of distinct average electronic couplings. Thus, it was possible to recover the exponential distance dependence expected from ET theory. These results pave the way for gaining further insight into the parameters that control protein electron transfer.

Electron-Transfer Rate in Potential-Modulated Redox Reactions with Electro-Active Optical Waveguides

“…Impedance measurements where data is collected over a range of modulation frequencies have proven useful for determination of time constants present in electrochemical processes. Usually electron-transfer kinetics of redox species needs be investigated under a variety of interfacial conditions [1][2][3] and electrochemical impedance spectroscopy have been widely deployed for those tasks. [4][5][6][7] However, electrochemical techniques based purely on electrical measurements invariably exhibit inadequate sensitivity to detect small faradaic currents from low concentrations of redox species on the electrode surface.…”

Orthogonal Translation Meets Electron Transfer: In Vivo Labeling of Cytochrome c for Probing Local Electric Fields

“…The essential role of cyt c in the chemistry of life and cell viability is well established:i tp articipates in fundamentalb iological processes such as respiration, photosynthesis, apoptosis, detoxificationa nd gas sensing. [3,5] Nonetheless, aq uantitative description of how local electric fields control the ET reaction mechanism is still missing. In particular,t he electron transfer (ET) functiono fc yt c has been studied in detail both for the protein in solution, in complex with its natural reaction partners, and in electrochemical environments.…”

“…When varying the scan rate, peak current increases linearly,t hus demonstrating that cyt c remains bound to the electrode surface ( Figure S7). [5,9,20] Alternatively, the redoxp otential change might be ac onsequence of ac hange in electrostatics due to introducing an additional dipole moment by the F! [19] The variant pCNF-cyt c displayed aslightly lower midpoint potentialo fa bout + +40 mV.T he shift in redoxp otential might be the result of as ubtle structuralp erturbation in the heme pocket (too small to be detected in the UV-visible and CD spectra).…”

“…In particular,t he electron transfer (ET) functiono fc yt c has been studied in detail both for the protein in solution, in complex with its natural reaction partners, and in electrochemical environments. [1,[3][4][5] Electrochemicald evices are particularly instructive as, upon coating the workinge lectrode with membrane models, one can mimic the natural reaction conditions at the mitochondrial membrane where the physiological redox process takes place. Thereby it was shown that high local electric fieldsa re likely to affect the interplay between protein dynamics and ET and, hence,t he efficiency of the redox process.…”

“…Thereby it was shown that high local electric fieldsa re likely to affect the interplay between protein dynamics and ET and, hence,t he efficiency of the redox process. [3,5] Nonetheless, aq uantitative description of how local electric fields control the ET reaction mechanism is still missing. To fill this gap, local electric field strengths can be determined by exploiting the vibrational Starkeffect (VSE), which requires the incorporating of an appropriate reporter group (e.g.,n itrile, carbonyl, azide) site-specifically into the protein.…”