Approach to Interfacial and Intramolecular Electron Transfer of the Diheme Protein Cytochrome c 4 Assembled on Au(111) Surfaces Qijin Chi,* ,† Jingdong Zhang, † Taner Arslan, † Lotte Borg, † Gert W. Pedersen, † Hans E. M. Christensen, † Renat R. Nazmudtinov, ‡ and Jens Ulstrup* ,† Department of Chemistry and Nano•DTU, Technical UniVersity of Denmark, KemitorVet, Building 207, DK-2800 Kongens Lyngby, Denmark, and Kazan State Technological UniVersity, 420015 Kazan, Republic of Tatarstan, Russian Federation ReceiVed: January 25, 2010; ReVised Manuscript ReceiVed: March 13, 2010 Intramolecular electron transfer (ET) between metal centers is a core feature of large protein complexes in photosynthesis, respiration, and redox enzyme catalysis. The number of microscopic redox potentials and ET rate constants is, however, prohibitive for experimental cooperative ET mapping, but two-center proteins are simple enough to offer complete communication networks. At the same time, multicenter redox proteins operate in membrane environments where conformational dynamics may lead to gated ET features different from conditions in homogeneous solution. The bacterial respiratory diheme protein Pseudomonas stutzeri cytochrome c 4 has been a target for intramolecular, interheme ET. We report here voltammetric and in situ scanning tunneling microscopy (STM) data for P. stutzeri cyt c 4 at single-crystal, atomically planar Au(111)- electrode surfaces modified by variable-length ω-mercapto-alkanoic carboxylic acids. As evidenced by in situ STM, the strongly dipolar protein is immobilized in a close to vertical orientation at this surface with the positively charged high-potential heme domain adjacent to the electrode. This orientation gives asymmetric voltammograms with two one-ET peaks in the cathodic direction and a single two-ET peak in the anodic direction. Intramolecular, interheme ET with high, 8,000-30,000 s -1 , rate constants is notably an essential part of this mechanism. The high rate constants are in striking contrast to ET reactions of P. stutzeri cyt c 4 with small reaction partners in homogeneous solution for which kinetic analysis clearly testifies to electrostatic cooperative effects but no intramolecular, interheme ET higher than 0.1-10 s -1 . This difference suggests a strong gating feature of the process. On the basis of the three-dimensional structure of P. stutzeri cyt c 4 , gating is understandable due to the through-space, hydrogen-bonded electronic contact between the heme propionates which is highly sensitive to environmental configurational fluctuations. 1. Introduction Biological electron transfer (ET) function in photosynthetic reaction centers, 1,2 respiratory complexes such as cytochrome c oxidase, 3-5 and redox enzymes 6-10 is controlled by large protein complexes with several transition metal centers. Com- munication between the membrane bound complexes is by small mobile ET carriers such as cytochrome c, plastocyanine, or quinones. The electronic conductivity through the protein complexes is determined by thermodynamic properties, say equilibrium redox potentials, coupling of the redox centers to the protein matrix and aqueous/membrane interfacial environ- ment, and the relative distance and orientation of the ET centers. Two other issues regarding long-range multi-ET “hopping” in redox (metallo)proteins require attention. The large number of microscopic interactions in multicenter redox proteins 11,12 is, first, prohibitive for complete microscopic thermodynamic and ET reactivity mapping with “cooperativity” included. Prototype two-center redox metalloproteins offer, however, complete mapping where the respiratory bacterial diheme protein cyto- chrome c 4 from Pseudomonas stutzeri has been a central target protein. 13-15 Figure 1 shows a three-dimensional structural representation of this protein, along with monoheme cytochrome c as a reference. The surface charge distribution and heme centers of P. stutzeri cyt c 4 are shown in Figure 2. Thermody- namic, 13-16 folding, 17 spectroscopic, 13,18,19 and ET kinetic properties 13,14,20,21 are also available. The preparation of the particular nuclear configuration in which ET occurs can, second, be critical. “Gated” ET is a notion used for this phenomenon 26 that implies expenditure of ad- ditional activation free energy in return for a more facile electronic transmission coefficient. This feature is important when ET is mediated in part by through-space links and particularly if ET is accompanied by proton transfer (PT), as the latter is spatially much more strongly confined than ET. The electronic transmission coefficient of intramolecular ET via a through-space link could thus assume widely different values in different environments such as bulk solution or in membrane or in interfacial electrochemical environment. The following observations are important when gated in- tramolecular ET between the two heme groups in P. stutzeri cyt c 4 is addressed: 13 • The 190-residue protein is organized in two almost equal- size globular domains, each with a heme group and connected by a 12-residue peptide chain. 22,25 The protein is strongly dipolar with excess negative and positive charge (pH 7) of the N- and C-terminal domain, respectively. This property is crucial for the electrostatic immobilization of the protein in * Corresponding authors. Phone: +45 252032 (Q.C.); +45 252359 (J.U.). Fax: +45 883136 (Q.C.); +45 883136 (J.U.). E-mail: cq@kemi.dtu.dk (Q.C.); ju@kemi.dtu.dk (J.U.). † Technical University of Denmark. ‡ Kazan State Technological University. J. Phys. Chem. B 2010, 114, 5617–5624 5617 10.1021/jp1007208  2010 American Chemical Society Published on Web 04/01/2010