scientific adviser

Csaba BAGYINKA scientific adviser
Alajos BÉRCZI scientific adviser
Gábor Miklós STEINBACH research associate
Zsolt SZEGLETES research associate
Attila Gergely VÉGH research associate


Electron transfer in proteins

Cytochromes are heme containing proteins, which carry out diverse physiological functions, such as electron transfer (ET), an important process in the energy metabolism of living cells. Intraprotein and interprotein ET is mediated by the protein matrix, as described by the Marcus theory. We have used a photoactive redox compound, thiouredopyrene-trisulfonate (TUPS) [1], invented by prof. A. Kotlyar (Tel Aviv University), to covalently label various lysine or engineered cysteine residues on the surface of mitochondrial cytochrome c (cytc), in order to study the kinetics of ET by multichannel flash-photolysis spectroscopy. We have introduced chemometric methods to analyze the spectrotemporal data matrices to characterize the heterogeneous ET kinetics observed in this system as well as in the complex of cytc and cytochrome c oxidase. We have identified optimal ET routes and explained the heterogeneous nature of the ET by molecular dynamics calculations [2]. We have also provided site directed surface mutants of cytc to the P. Hildebrandt group at TU Berlin. As a result of this collaboration it was possible to study the intricate interplay of protein dynamics, governed by surface electric field, and the distance-dependent electron tunneling on cytc and self-assembled monolayer coated electrodes, modeling the mitochondrial inner membrane – cytc interaction [3,4].

Recently, we have extended our studies to members of the cytochrome b561 (Cyt-b561) protein family (2 heme, 6 transmembrane helix, ascorbate reducible electron transporters from a wide variety of organisms). There is only sporadic information about the physiological role of these proteins and little is known about the structural background of their ET function. Together with A. Bérczi we have spectrally characterized the interaction of various electron donor molecules and the tonoplast and a putative tumor suppressor Cyt-b561. In the two proteins the low and high affinity hemes have opposite absorption spectral properties indicating different interactions with the heme binding pockets. Double mutation of the axial ligands of one heme in the tonoplast protein has shown that the high-potential heme site is situated at the cytoplasmic side of the membrane and allowed the unambiguous differentiation between two models on the heme localization in Cyt-b561 proteins [5-7]. The direction of the transmembrane ET is opposite to the midpoint redox potential difference between the two hemes and is, therefore, driven by either the membrane potential or by other thermodynamically coupled downhill reaction(s). Homology modeling of a number of Cyt-b561 proteins using the only available crystal structure allowed us to conclude that no amino acid is conserved in the region separating the two hemes (most conserved amino acids contribute either to the heme pockets or to the substrate binding sites). Therefore there seems to be no other structural requirement for efficient ET between the hemes than a sufficient packing density of the protein matrix.

Structure of a Cyt-b561 with the conserved amino acids in red (left) and the calculated dominant ET pathway between the two hemes (right).

Proteins in photonic crystals

In recent years we have collaborated with French and Mexican groups and with the Institute of Medical Physics and Informatics of Szeged University in the field of biophotonics and bioelectronics, based on porous silicon (PSi) infiltrated with biomacromolecules. PSi materials can be relatively easily produced by programmed electrochemical etching on the surface of commercial silicon wafers. Appropriate periodic tuning of the porosity results in structures with periodic refractive index changes, i.e. photonic crystals. PSi material is characterized also by a very high active surface area and the possibility of physical or chemical functionalization. We have incorporated glucose oxidase, cytc, solubilized, monomeric bacteriorhodopsin and photosynthetic reaction center proteins into PSi photonic crystals. We have visualized the inner structure of such materials in a multiphoton microscope setup utilizing the nonlinear optical response – second harmonic generation and two-photon luminescence – of the embedded proteins [8,9]. We are also exploring the possibility of producing PSi working electrodes for protein film voltammetry of redox proteins. Cytc alone and in complex with the photosynthetic reaction center are prime candidates as electron transferring proteins at the current stage of these studies [10,11]. The electronic interaction of these or other proteins with the semiconductor silicon material is an interesting aspect of this project with potential future applications. The photonic properties of various PSi architectures and the influence of the embedded biomacromolecules thereon is another area of potential future applications in e.g. biomolecular detection. Similarly promising is the selective high affinity of certain peptides towards the Si surface, a subject we study within a French-Hungarian collaboration [12].

3D structure of a porous Si microcavity photonic crystal visualized by multiphoton microscopy.

Selected publications

[1] Kotlyar, A.B., Borovok, N., Khoroshyy, P., Tenger, K. and Zimányi. L. (2004) Redox photochemistry of thiouredopyrenetrisulfonate. Photochem. Photobiol. 79(6): 489-493

[2] Tenger, K., Khoroshyy, P., Leitgeb, B., Rákhely, G., Borovok, N., Kotlyar, A., Dolgikh, D.A. and Zimányi, L. (2005) Complex kinetics of the electron transfer between the photoactive redox label TUPS and the heme of cytochrome c. J. Chem. Inf. Mod. 45(6):1520-1526

[3] Schkolnik, G., Utesch, T., Salewski, J., Tenger, K., Millo, D., Kranich, A., Zebger, I., Schulz, C., Zimányi, L., Rákhely, G., Mroginski, M.A., Hildebrandt, P. (2011) Mapping local electric fields in proteins at biomimetic interfaces. ChemComm., 48:70-72

[4] Alvarez-Paggi, D., Meister, W., Kuhlmann, U., Weidinger, I., Tenger, K., Zimányi, L., Rákhely, G., Hildebrandt, P. and Murgida, D.H. 2013. Disentangling electron tunneling and protein dynamics of cytochrome c through a rationally designed surface mutation. J. Phys. Chem. B. 117(20):6061–6068

[5] Desmet, F., Bérczi, A., Zimányi, L., Asard, H. and Van Doorslaer, S. (2011) Axial ligation of the high-potential heme center in an Arabidopsis cytochrome b561. FEBS Lett. 585:545–548

[6] Bérczi, A., Zimányi, L. and Asard, H. 2013. Dihydrolipoic acid reduces cytochrome b561 proteins. Eur. Biophys. J. 42:159–168

[7] Bérczi, A. and Zimányi, L. 2014. The trans-membrane cytochrome b561 proteins: structural information and biological function. Curr. Protein Pept. Sci. 15:745-760

[8] Martin, M., Palestino, G., Cloitre, T., Agarwal, V., Zimányi, L. and Gergely, Cs. (2009) Three dimensional spatial resolution of the nonlinear photoemission from biofunctionalized porous silicon microcavity. Appl. Phys. Lett. 94:223313-1-3

[9] Palestino, G., Martin, M., Agarwal, V., Legros, R., Cloitre, T., Zimányi, L. Gergely, Cs. (2009) Detection and light enhancement of glucose oxidase adsorbed on porous silicon microcavities. Phys. Stat. Solidi C 6(7):1624–1628

[10] Hajdu, K., Gergely, C., Martin, M., Cloitre, T., Zimányi, L., Tenger, K., Khoroshyy, P., Palestino, G., Agarwal, V., Hernádi, K., Németh, Z. and Nagy, L. (2012) Porous silicon/photosynthetic reaction center hybrid nanostructure. Langmuir 28:11866-11873

[11] Márquez, J., Cházaro-Ruiz, L.F., Zimányi, L. and Palestino, G. (2014) Immobilization strategies and electrochemical evaluation of porous silicon based cytochrome c electrode. Electrochim. Acta 140:550-556

[12] Pápa, Z., Ramakrishnan, S.K., Martin, M., Cloitre, T., Zimányi, L., Márquez, J., Budai, J., Tóth, Z. and Gergely, C. (2016). Interactions at the peptide/silicon surfaces: Evidence of peptide multilayer assembly. Langmuir 32(28): 7250–7258