Kinetic isotope effects reveal an ice‐like and a liquid‐phase‐type intramolecular proton transfer in bacteriorhodopsin

137

144

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...

Section: Discussionsupporting

confidence: 84%

Structures and spectral signatures of protonated water networks in bacteriorhodopsin

Mathias

Marx

2007

137

144

“…The resulting barrier was determined to be approximately 6 RT adjacent to membranes made of carbon nanotubes (20). This observation does not explain why the barrier adjacent to GMO membranes (8.8-10 RT) is smaller than that in the vicinity of DPhPC membranes (12)(13)(14)(15)(16).…”

Section: Discussionmentioning

confidence: 87%

Protons migrate along interfacial water without significant contributions from jumps between ionizable groups on the membrane surface

Springer

Hagen²,

Cherepanov³

et al. 2011

100

140

Kinetics of fluorescence changes on top of three different lipid bilayers due to lateral proton migration. The observation area was located at a distance of 70 μm from the area of proton release. Because all FPE molecules are surrounded by DPhPC or DPhPE molecules, they are anticipated to accept protons, which are released from these molecules. GMO does not possess ionizable moieties so that in case of two-dimensional diffusion, proton release from one FPE molecule seems to be required before the next FPE molecule may pick up the proton. Despite the huge differences in proton release rates from the different lipids, τ max for all three lipid bilayers was similar. That is, lateral proton diffusivity is independent of the choice of the lipid. The buffer contained 0.1 mM Capso (pH 9.0) and 100 mM NaCl. The F1 Wv/+ mice were then backcrossed to A/J mice for 10 generations, creating A/J N10 Wv/+ mice. These were then crossed to B6 Wv/+ mice to create F1 wildtype and F1 Wv/Wv mice for study. A/J mice had an increased airway resistance compared to B6 mice, and F1 mice had a naïve AHR phenotype equivalent to their parental A/J strain. F1 Wv/Wv mice displayed an airway resistance similar to normoresponsive B6 mice. Values represent mean ± SE, n = at least 10 in each group.

“…In this system only the L 3 M transition, which involves proton transfer from the Schiff base to Asp-85, and the decay of the O state, which is associated with deprotonation of Asp-85, display large KIE of 5-10. All other transitions, which are associated with proton transfer across the protein display much smaller KIE of Ϸ2 (41,45,46). Thus, also in bacteriorhodopsin, a large KIE is associated only with proton transfer across the pumping element.…”

Section: Discussionmentioning

confidence: 97%

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

Salomonsson

Faxén

Ädelroth

et al. 2005

In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O2. The reduction of O2, accompanied by the uptake of substrate protons to form H2O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H2O the uptake and release reactions occur with the same rates, in D2O the substrate and pumped protons are taken up first (D Х 200 s, ''peroxy'' to ''ferryl'' transition) followed by a significantly slower proton release to the other side of the membrane (D Х 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation.heme-copper oxidases ͉ electron transfer ͉ membrane protein ͉ respiratory chain I n membrane-bound proton pumps the endergonic proton translocation across the membrane is driven by an exergonic reaction, e.g., electron transfer from a low-potential donor to a high-potential acceptor. The protons are translocated through proton-transfer pathways, typically composed of networks of hydrogen-bonded protonatable and polar amino acid side chains and bound water molecules, that span the transmembrane part of the protein. To prevent short-circuiting of the electrochemical gradient it is crucial that there is never simultaneous access (i.e., a direct contact) to the two membrane sides. One way to control the proton access is to alter the position of e.g., an amino acid side chain such that the group exchanges protons rapidly with either one side or the other side of the membrane (but never both sides simultaneously), defining an input and an output state (for review, see refs. 1-6). Proton pumping would be accomplished, for example, if the reaction that drives proton translocation is energetically coupled to changes in the pK a value of the protonatable group such that it has a high pK a in the input state and a low pK a in the output state. In the discussion below, we will refer to such a group as a ''pumping element.'' Cytochrome c oxidase (CcO), which belongs to the hemecopper oxidase superfamily, is an example of a redox-driven proton pump (for review, see refs 4, 5, and 7-12). The CcOs are found in the cytoplasmic membrane in bacteria or the inner membrane of mitochondria, where they catalyze the reduction of molecular oxygen to water. The electrons are donated by...