Structural Biology DOI: 10.1002/anie.201001243 Directional Proton Transfer in Membrane Proteins Achieved through Protonated Protein-Bound Water Molecules: A Proton Diode** Steffen Wolf, Erik Freier, Meike Potschies, Eckhard Hofmann, and Klaus Gerwert* Dedicated to Professor Manfred Eigen The key function of energy-transducing membrane proteins is the creation of a proton gradient by directional proton transfer. The role of protein-bound water molecules herein is not fully understood, as X-ray diffraction analysis has resolved the positions of oxygen, but not of hydrogen atoms in such protein–water complexes. Here we show, now time- resolved at atomic resolution, how a membrane protein achieves directional proton transfer via protein-bound water molecules in contrast to random proton transfer in liquid water. A combination of X-ray structure analysis, time- resolved FTIR spectroscopy, and molecular dynamics (MD) simulations elucidates how directionality is achieved. Using the proton-pump bacteriorhodopsin as the paradigm, we show how controlled conformational changes of few amino acid residues rearrange preordered water molecules and induce directional proton transfer. This mechanism is analo- gous to an electronic diode: a “proton diode”. According to the chemiosmotic theory, the creation of a proton gradient in photosynthesis [1] and oxidative phosphor- ylation [2–4] by means of directional proton transfer is the key step for energy transduction in living cells. ATPases use this proton gradient to produce ATP, the fuel for life. In contrast to this directional mechanism in proteins, proton transfer in liquid water is random. [5, 6] Bacteriorhodopsin (bR), a protein that belongs to the microbial rhodopsin family, [7, 8] achieves this directional proton transfer by a light-driven proton- pumping mechanism. Like other microbial rhodopsins, bR exhibits a structural motif of seven transmembrane a-helices and a retinal chromophore covalently bound to a lysine through a protonated Schiff base. The light-induced retinal isomerization from all-trans in the ground state (BR) to the 13-cis conformer drives bR through a photocycle with intermediates named J, K, L, M, N, and O in order of their appearance. [8] During the L to M transition, the protonated Schiff base (C =NH + ), the central proton-binding site, deprotonates and protonates its counterion Asp85 [9] (step 1 in Figure 1 a). Protonation of Asp85 breaks its salt bridge to Arg82, which then moves towards Glu194/Glu204 (step 2). The orientation of Arg82 depends on the protonation state of Asp85. [10] The arginine movement destabilizes a protonated water cluster between Arg82, Glu194, and Glu204 (step 3 in Figure 1 a), and a proton is released to the bulk. [11, 12] However, the detailed nature of the proton-release group is still under debate. QM/MM simulations of the proton- release group propose a shared proton between Glu194 and Glu204, [13] a Zundel cation with two water molecules (H 5 O 2 + ), [14] or an asymmetric Eigen cation of four water molecules (H 9 O 4 + ). [15] From time-resolved FTIR experiments with site-directed mutations around the protonated water cluster and H/D-exchange experiments we have concluded that the proton-release group forms a protonated water cluster, most likely an asymmetric Eigen ion as shown in Figure 1 a in purple. [12] Glu194 and Glu204 are clearly deprotonated in the bR ground state. [11] This experimental result was recently confirmed by Lórenz-Fonfría et al. [16] Nevertheless, the exact nature of the protonated water cluster and the release mechanism has still to be determined. Here, we used X-ray structure analysis to determine the positions of the water oxygen atoms and FTIR difference spectroscopy to determine the dynamics of the corresponding water hydrogen atoms. The proton release to the bulk in the L [*] Dr. S. Wolf, [+] E. Freier, [+] Dr. M. Potschies, [$] Prof. Dr. E. Hofmann, Prof. Dr. K. Gerwert Lehrstuhl für Biophysik, Ruhr-University Bochum Universitätsstrasse 150, 44780 Bochum (Germany) Fax: (+ 49) 234-321-4238 E-mail: gerwert@bph.rub.de Homepage: http://www.bph.rub.de Dr. S. Wolf, [+] Prof. Dr. K. Gerwert Department of Biophysics CAS–Max-Planck Partner Institute for Computational Biology Shanghai Institutes for Biological Sciences 320 Yue Yang Road, 200031 Shanghai (P.R. China) [ $ ] Current address: Lehrstuhl für Biophysik, Universität Konstanz (Germany) [ + ] These authors contributed equally to this work. [**] We thank R. E. Moritz and D. Onidas for improving the English of the manuscript, Q. Cui, D. Bashford, and J. Schlitter for helpful discussions, G. Smuda for the preparation of bR mutants, and the NIC Jülich (project no. hbo26) and the RRZK Köln for providing computing time. We thank the beamline staff at the Swiss Light Source and at the European Synchrotron Radiation Source for help during data collection. Atomic coordinates and structure factors for the reported crystal structures have been deposited at the Protein Data Bank under access codes 2WJK (E204D) and 2WJL (E194D). This work was supported by the Deutsche Forschungsgemeinschaft (DFG GE-599/12-3) and by the Ruhr-University Research School. Molecular figures were prepared with PyMOL. Author contribu- tions: S.W.: MD simulations, structure analysis, and sequence alignment; E.F.: FTIR measurements and structure analysis; M.P. and E.H.: protein crystallization and solution of the E194D and E204D crystal structures; K.G.: design and organization of the project; S.W., E.F., and K.G.: manuscript preparation. Supporting information for this article (including details of protein purification, FTIR measurements, X-ray crystallography, and MD simulations) is available on the WWW under http://dx.doi.org/10. 1002/anie.201001243. A ngewandte Chemi e 6889 Angew. Chem. Int. Ed. 2010, 49, 6889 –6893  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim