A local area network of protonated water molecules.

Water is the major component of all living cells, actually it composes 70% of our bodies. Its properties, very unusual when compared to other solvents, have made possible the life that we know. Water molecules spontaneously dissociate into a hydronium (H3O þ) and a hydroxide ion (OH). This autoprotolysis is a very rare event with the average lifetime of a single H2O molecule being ;14 h (Eigen, 1964). However, not all of the H2O molecules in our body are what we call liquid water. H2O molecules can be tightly bound to biological material and are occluded in proteins where they are often involved in catalytic reactions. The membrane protein bacteriorhodopsin (bR) accommodates several of such water-filled cavities (Dencher et al., 2000). Their participation in the lightdriven proton translocation, which is the functional task of this molecular machine, is intensively studied. A cavity close to the extracellular membrane surface accommodates a local area network (LAN) of hydrogenbonded water molecules and amino acid side chains. This LAN houses an excess proton, which is released after photoexcitation of bacteriorhodopsin. Over the recent years, the group of K. Gerwert has specifically addressed the role of this LAN by time-resolved Fourier transform infrared spectroscopy. In this issue of the Biophysical Journal, Garczarek et al. (2004) critically gauged the characteristic infrared (IR) spectroscopic signatures of the excess proton within this LAN. As an excellent scientific practice, they solved the controversy about the nature of the spectral changes in collaboration with the group of M. El-Sayed. Continuum absorbance changes due to the release of the excess proton could be clearly distinguished from photothermal heating artifacts of bR. This is a very important result since the concept of the continuum bands might be applicable to other proton translocating proteins as well. Considering that almost all known enzymatic mechanisms involve proton transfers, these are issues of major significance for understanding protein function in general. Besides the role per se, the transfer of protons leads to the redistribution of charges in a protein. By these electrostatic means, structural changes of the protein are triggered that may induce changes in affinity to ligands or to interacting proteins. As a prerequisite for proton translocation, a proton-conducting wire must exist, made of water molecules and/or ionizable amino acid side chains. The remarkably fast proton transfer in water (diffusion constant DHþ 1⁄4 9.3 · 10 m/s) can be related to the Grotthuss mechanism, where the charge of the proton is displaced along the hydrogen-bonded network of water molecules rather than the mass. Such a mechanism is effective only when the involved hydrogen bonds are easy to break and the cleavage of a single hydrogen bond of liquid water requires only ;10 kJ/mol. The energetics of proton transfer can be envisaged as a double-well potential where the proton is transferred from a donor (left well in Fig. 1) to the acceptor (right well). The key for fast proton transfer lies in the height of the intermittent barrier. Lowering the barrier by bringing the donor and acceptor molecules in appropriate distance and orientation will accelerate proton transfer. The efficiency of proton transfer is determined by the potential level (free energy) of the donor and the acceptor, respectively. For proton transfer in water, the double-well potential is symmetrical and the barrier is low (Fig. 1). Thus, protons can be rapidly transferred between donor and acceptor. It was Georg Zundel who demonstrated that this ‘‘large proton polarizability’’ gives rise to intense continua in the IR spectra (Zundel, 1992). For a heterogeneous proton-conducting chain in a protein, e.g., like that of the extracellular LAN of bR, a series of such potentials may exist for the protonatable groups (peptide backbone, amino acid side chains, and water molecules) where the barrier for proton transfer is low and the lowest potential well determines the localization of the proton. Putting energy into the system, e.g., by light absorption in photosynthetic proteins, will shift the potential wells with respect to each other and proton transfer ensues. If the last member of the chain has the lowest potential, the proton gets trapped in this well. Efficient proton translocation is deducible from the occurrence of broad negative bands in the IR difference spectrum because the proton with its large polarizability is lost. Protons, although not as small from the standpoint of mass as electrons, are sufficiently light for treating their properties by quantum mechanics. Marcus theory, which has been extremely useful for our current understanding of electron transfer in biological systems, can also be applied to advance our knowledge of the possible pathways for proton transfer (Silverman, 2000). The thermal de Broglie wavelength of the proton is 1.5 Å, which compares well with the distances of proton transfer reactions. An intriguing consequence is that protons may tunnel from a proton donor to the acceptor, i.e., they do not pass the transition state but rather cross the potential energy barrier (Fig. 1). The Submitted August 3, 2004, and accepted for publication August 6, 2004.