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Long-distance proton transfer with a break in the bacteriorhodopsin active site.

ABSTRACT: Bacteriorhodopsin is a proton-pumping membrane protein found in the plasma membrane of the archaeon Halobacterium salinarium. Light-induced isomerization of the retinal chromophore from all-trans to 13-cis leads to a sequence of five conformation-coupled proton transfer steps and the net transport of one proton from the cytoplasmic to the extracellular side of the membrane. The mechanism of the long-distance proton transfer from the primary acceptor Asp85 to the extracellular proton release group during the O --> bR is poorly understood. Experiments suggest that this long-distance transfer could involve a transient state [O] in which the proton resides on the intermediate carrier Asp212. To assess whether the transient protonation of Asp212 participates in the deprotonation of Asp85, we performed hybrid Quantum Mechanics/Molecular Mechanics proton transfer calculations using different protein structures and with different retinal geometries and active site water molecules. The structural models were assessed by computing UV-vis excitation energies and C=O vibrational frequencies. The results indicate that a transient [O] conformer with protonated Asp212 could indeed be sampled during the long-distance proton transfer to the proton release group. Our calculations suggest that, in the starting proton transfer state O, the retinal is strongly twisted and at least three water molecules are present in the active site.

SUBMITTER: Phatak P

PROVIDER: S-EPMC2746972 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Long-distance proton transfer with a break in the bacteriorhodopsin active site.

Phatak Prasad P Frähmcke Jan S JS Wanko Marius M Hoffmann Michael M Strodel Paul P Smith Jeremy C JC Suhai Sándor S Bondar Ana-Nicoleta AN Elstner Marcus M

Journal of the American Chemical Society 20090501 20

Bacteriorhodopsin is a proton-pumping membrane protein found in the plasma membrane of the archaeon Halobacterium salinarium. Light-induced isomerization of the retinal chromophore from all-trans to 13-cis leads to a sequence of five conformation-coupled proton transfer steps and the net transport of one proton from the cytoplasmic to the extracellular side of the membrane. The mechanism of the long-distance proton transfer from the primary acceptor Asp85 to the extracellular proton release grou ...[more]

PMID: 19405533

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Project description:To test the effect of varying the proton donor-acceptor distance in proton-coupled electron transfer (PCET) reactions, the oxidation of a bicyclic amino-indanol (2) is compared with that of a closely related phenol with an ortho CPh(2)NH(2) substituent (1). Spectroscopic, structural, thermochemical, and computational studies show that the two amino-phenols are very similar, except that the O···N distance (d(ON)) is >0.1 Å longer in 2 than in 1. The difference in d(ON) is 0.13 ± 0.03 Å from X-ray crystallography and 0.165 Å from DFT calculations. Oxidations of these phenols by outer-sphere oxidants yield distonic radical cations (•)OAr-NH(3)(+) by concerted proton-electron transfer (CPET). Simple tunneling and classical kinetic models both predict that the longer donor-acceptor distance in 2 should lead to slower reactions, by ca. 2 orders of magnitude, as well as larger H/D kinetic isotope effects (KIEs). However, kinetic studies show that the compound with the longer proton-transfer distance, 2, exhibits smaller KIEs and has rate constants that are quite close to those of 1. For example, the oxidation of 2 by the triarylamminium radical cation N(C(6)H(4)OMe)(3)(•+) (3a(+)) occurs at (1.4 ± 0.1) × 10(4) M(-1) s(-1), only a factor of 2 slower than the closely related reaction of 1 with N(C(6)H(4)OMe)(2)(C(6)H(4)Br)(•+) (3b(+)). This difference in rate constants is well accounted for by the slightly different free energies of reaction: ?G° (2 + 3a(+)) = +0.078 V versus ?G° (1 + 3b(+)) = +0.04 V. The two phenol-amines do display some subtle kinetic differences: for instance, compound 2 has a shallower dependence of CPET rate constants on driving force (Brønsted ?, ? ln(k)/? ln(K(eq))). These results show that the simple tunneling model is not a good predictor of the effect of proton donor-acceptor distance on concerted-electron transfer reactions involving strongly hydrogen-bonded systems. Computational analysis of the observed similarity of the two phenols emphasizes the importance of the highly anharmonic O···H···N potential energy surface and the influence of proton vibrational excited states.

Project description:The cytoplasmic surface of the BR (initial) state of bacteriorhodopsin is characterized by a cluster of three carboxylates that function as a proton-collecting antenna. Systematic replacement of most of the surface carboxylates indicated that the cluster is made of D104, E161, and E234 (Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281-4292), yet the BR state is a resting configuration; thus, its proton-collecting antenna can only indicate the presence of its role in the photo-intermediates where the protein is re-protonated by protons coming from the cytoplasmic matrix. In the present study we used the D96N and the triple (D96G/F171C/F219L) mutant for monitoring the proton-collecting properties of the protein in its late M state. The protein was maintained in a steady M state by continuous illumination and subjected to reversible pulse protonation caused by repeated excitation of pyranine present in the reaction mixture. The re-protonation dynamics of the pyranine anion was subjected to kinetic analysis, and the rate constants of the reaction of free protons with the surface groups and the proton exchange reactions between them were calculated. The reconstruction of the experimental signal indicated that the late M state of bacteriorhodopsin exhibits an efficient mechanism of proton delivery to the unoccupied-most basic-residue on its cytoplasmic surface (D38), which exceeds that of the BR configuration of the protein. The kinetic analysis was carried out in conjunction with the published structure of the M state (Sass, H., G. Büldt, R. Gessenich, D. Hehn, D. Neff, R. Schlesinger, J. Berendzen, and P. Ormos. 2000. Nature. 406:649-653), the model that resolves most of the cytoplasmic surface. The combination of the kinetic analysis and the structural information led to identification of two proton-conducting tracks on the protein's surface that are funneling protons to D38. One track is made of the carboxylate moieties of residues D36 and E237, while the other is made of D102 and E232. In the late M state the carboxylates of both tracks are closer to D38 than in the BR (initial) state, accounting for a more efficient proton equilibration between the bulk and the protein's proton entrance channel. The triple mutant resembles in the kinetic properties of its proton conducting surface more the BR-M state than the initial state confirming structural similarities with the BR-M state and differences to the BR initial state.

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Long-distance proton transfer with a break in the bacteriorhodopsin active site.

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Long-distance proton transfer with a break in the bacteriorhodopsin active site.

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