Cytochrome C-Calixarene Crystals on Electrodes: Electron Transfer Between Defined Redox Sites

Abstract

Cytochrome c (cyt c, 13 kDa) is a well-characterized redox protein[1] which has been used extensively as model protein and for the construction of artificial signal chains in biosensors.[2,3] Often this involves immobilization of the cationic cyt c on gold electrodes modified by self-assembled monolayers (SAM) of packed anionic alkanethiols.[4] One route to overcome the immobilisation and addressability of a monolayer of the redox protein is the layer-by-layer (LbL) adsorption technique.[6,7] Via the LbL heterogeneous multilayer assemblies have been constructed with cyt c and anionic “glues” such as DNA, silica nanoparticles, or polyaniline sulfonic acid (PASA).[6,7] The electrochemical response increased with the number of layers as a result of direct interprotein electron transfer between the cyt c molecules. The epitome of highly concentrated and ordered protein assemblies with potential biological relevance are protein crystals.[8] Evidences of possible electron transfer in redox protein crystals have been reported.[9,10] Remarkably, so far there has been only one report of direct electrochemical characterization of protein crystals.[11] In this study, cyt c was co-crystallized with p-sulfonato-calix[4]arene (sclx4) on gold electrodes. Sclx4 is a bowl-shaped molecular glue that can be used for protein crystallization.[12] The Sclx4 shares some chemical properties with PASA. Notably, similar charge–charge interactions between the polyanion and the cationic cyt c may drive protein complexation and assembly.[6,12] The key difference between the two systems is that the exact position of each protein is defined in the protein crystal formed with sclx4, while the multilayer assembly formed with PASA comprises a heterogeneous mixture of protein–polymer and protein–protein contacts. During the investigation conditions have been identified to ensure crystal formation directly on a modified gold electrode and stability for direct electrochemical characterization [13]. The crystal electrodes were characterized by cyclic voltammetry and the crystal coverage has been quantified microscopically and correlated with the electrochemical data. Exceptionally high concentrations of electroactive cyt c have been obtained. Furthermore the rate constant for electron self-exchange has been evaluated from scan rate dependent measurements. Electron transfer was found to occur through the crystal via interprotein ET between defined sites of the cyt c molecules. This proof-of-principle study reveals for the first time that the electron self-exchange properties of cyt c can be exploited for long-range electron transport in the solid state on electrodes. [1] H. B. Gray, J. R. Winkler, Biochim. Biophys. Acta Bioenerg. 2010, 1797, 1563. [2] R. Dronov, D. G. Kurth, H. Mçhwald, F. W. Scheller, F. Lisdat, Angew. Chem. Int. Ed. 2008, 47, 3000. [3] S. C. Feifel, R. Ludwig, L. Gorton, F. Lisdat, Langmuir 2012, 28, 9189. [4] S. Song, R. A. Clark, E. F. Bowden, M. J. Tarlov, J. Phys. Chem. B 1993, 97, 6564. [5] B. Ge, F. Lisdat, Anal. Chim. Acta 2002, 454, 53. [6] M. K. Beissenhirtz, F. W. Scheller, F. Lisdat, Anal. Chem. 2004, 76, 4665. [7] S. C. Feifel, F. Lisdat, J. Nanobiotechnol. 2011, 9:59. [8] P. B. Crowley, P. M. Matias, H. Mi, S. J. Firbank, M. J. Banfield, C. Dennison, Biochemistry 2008, 47, 6583. [9] S. A. Kang, B. R. Crane, Proc. Natl. Acad. Sci. USA 2005, 102, 15465. [10] C. Cavalieri, N. Bierman, M. D. Vlasie, O. Einsle, A. Merli, D. Ferrari, G. L. Rossi, M. Ubbink, Biochemistry 2008, 47, 6560. [11] F. Acosta, D. Eid, L. Marin-Gracia, B. A. Frontana-Uribe, A. Moreno, Cryst. Growth Des. 2007, 7, 2187. [12] R. E. McGovern, H. Fernandes, A. R. Khan, N. P. Power, P. B. Crowley, Nat. Chem. 2012, 4, 527. [13] Roise McGovern, S. C. Feifel, F. Lisdat, P. Crowley, Angew. Chem. Int. Ed. 2015, 54, 6356.