Structures and spectral signatures of protonated water networks in bacteriorhodopsin

Abstract

Networks of internal water molecules are thought to provide proton transfer pathways in many enzymatic and photosynthetic reactions. Extremely broad absorption continua observed in recent IR spectroscopic measurements on the photodriven proton pump bacteriorhodopsin (BR) suggest such networks may also serve as proton storage and release sites for these reactions. By combining electronic structure calculations with molecular mechanical force fields, we examine the dynamics and the resulting IR spectra of two protonated water networks, H ؉ ⅐(H2O)3 and H ؉ ⅐(H2O)4, in the release pocket of the initial state of BR, which possibly serve as proton donors to the extracellular surface. For both network sizes, topologically similar structures are found, which are anchored at residues E194 and E204 and stabilized by additional hydrogen bonds from neighboring protein side chains. These protonated water networks assume neither the classic Zundel nor Eigen motives but prefer wire-like topologies. Upon gauging calculated IR spectra of finite clusters with experimental gas-phase data, it is possible to link spectral features computed for these chain-like structures in the initial state of the BR photocycle to the measured absorption continua, in particular for the larger H ؉ ⅐(H2O)4 network. Furthermore, the free energy of proton dislocation along these chains is found to be within the range that is easily accessible at room temperature because of fluctuations.hydrogen-bonded networks ͉ hybrid molecular dynamics ͉ proton transport ͉ IR spectroscopy T he light-driven proton pump bacteriorhodopsin (BR) converts light energy to chemical energy by a vectorial proton transport against a membrane potential (1). In addition to belonging to the best-studied proton-permeable ion channels (2) as such and being a natural host for studying proton conducting water wires (3), BR is an important representative of the G protein-coupled seven-␣-helix receptor family (4). Thus, it comes as no surprise that this protein serves as a paradigmatic workhorse for both experiment and simulation. During its photocycle, protons are translocated to different positions within the transport channel, and a detailed dynamical picture is currently emerging for BR from the level of conformational changes to side-chain motion to individual atom displacements (5-9). In addition, biomolecular simulation strongly suggests the presence of locally mobile internal water molecules inside BR, which could easily serve as proton hosts and relays and thus establish a pathway from intracellular to extracellular space (10-12). A crucial step in this vectorial proton transfer is the actual release of a proton to the extracellular surface involving the so-called proton release group (XH). However, the very nature of XH is still a puzzling and disputed question.Whereas other initial as well as transient proton positions in BR have been quite well characterized (9), the terminal release group has withdrawn itself from identification for a long time. Recent IR spectroscopic meas...

Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

“…The continuum electrostatics calculations, however, were based on simple point-charge models and therefore are not conclusive; the close distances between Glu-194, Glu-204 and the relevant water molecules indicate that a quantum mechanical treatment of these residues is warranted for a quantitative consideration of the protonation pattern in this region. CarParrinello molecular dynamics (CPMD)-based quantum mechanical/molecular mechanical (QM/MM) simulations (6) found that a protonated water cluster in the PRG region generated a continuum IR band qualitatively consistent with the experimental observation, providing further support to the idea of involving water clusters rather than amino acid side chains as the proton storage site. However, the QM region included only the protonated water molecules and therefore did not allow proton transfer to the Glu residues.…”

“…Another important mechanistic issue concerns the identity of proton storage site(s), which is difficult to determine unambiguously because protons are invisible in the crystal structure of most biomolecules. Indeed, although titratable amino acids are the common suspect for proton storage sites, the unconventional possibility of storing proton in water clusters has also been raised in several recent studies (4)(5)(6)(7).…”

The vibrational spectra of protonated water clusters: A benchmark for self-consistent-charge density-functional tight binding

“…Although water molecules are well known to transiently facilitate proton transfers in many different settings, recent Fourier-transform infrared studies found evidence for the existence of long-lived protonated water cluster as the "proton storage site" in bacteriorhodopsin; 8,9 the interpretation seems to be supported by computational studies. 9,10 Recent simulation study of Xu et al also raised a similar possibility in cytochrome c oxidase. 11 To thoroughly examine these proposals, it is imperative to be able to compute reliable IR spectra for water clusters in different protonation states in a biomolecular environment.…”

“…The computational efficiency of SCC-DFTB makes it, in principle, an attractive choice in this context and complementary to calculations based on much more expensive QM methods that might suffer from convergence issues. 10 Although previous studies based on NMA indicate that SCC-DFTB overall gives favorable vibrational frequencies among semiempirical methods, 28,29 whether it is capable of capturing the spectroscopic signatures of various water clusters remains unknown. In this study, we carry out SCC-DFTB calculations for the IR spectra of various protonated water clusters in the gas phase and compare the results to experimental spectra, which only became available very recently.…”

“…Therefore, these characteristic signatures can be used to probe the possible existence and structure of a ͑protonated/ neutral͒ water network in the active site of enzymes. [8][9][10] …”