A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

Tittor, Jörg; Soell, Christa; Oesterhelt, Dieter; Butt, Hans‐Jürgen; Bamberg, Ernst

doi:10.1002/j.1460-2075.1989.tb08512.x

Cited by 194 publications

(158 citation statements)

References 38 publications

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“…Such behavior is strikingly different from that in the homologous E142Q mutant of NR, where the Schiff base reprotonation rate is not affected (6). Sodium azide, known to function as a proton shuttle in hydrophobic regions of haloarchaeal rhodopsins (23,24), restored the photocycle kinetics of the D150N mutant to rates comparable with the wild-type protein (Fig. 3d), similarly to the D96N mutant of BR.…”

Section: Resultsmentioning

confidence: 69%

Leptosphaeria rhodopsin: Bacteriorhodopsin-like proton pump from a eukaryote

Waschuk

Bezerra

Shi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

219

183

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Bacteriorhodopsin-like proteins provide archaea and eubacteria with a unique bioenergetic pathway comprising light-driven transmembrane proton translocation by a single retinal-binding protein.Recently, homologous proteins were found to perform photosensory functions in lower eukaryotes, but no active ion transport by eukaryotic rhodopsins was detected. By demonstrating lightdriven proton pumping in a fungal rhodopsin from Leptosphaeria maculans, we present a case of a retinal-based proton transporter from a eukaryote. This result implies that in addition to oxidative phosphorylation and chlorophyll photosynthesis, some lower eukaryotes may have retained the archaeal route of building an electrochemical transmembrane gradient of protons.proton transport ͉ sensory rhodopsins

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Section: Resultsmentioning

confidence: 69%

Leptosphaeria rhodopsin: Bacteriorhodopsin-like proton pump from a eukaryote

Waschuk

Bezerra

Shi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

219

183

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“…If the fatty acid is complementing the loss of the D132 carboxyl, or interacting at another site to improve proton access, this resembles the chemical rescue phenomenon observe with carboxylate mutants of other proton transferring membrane proteins. In mutated bacteriorhodopsin [14,15] and bacterial photosynthetic reaction center [16] activity can be restored by weak acid anions such as azide. These water soluble acids are required at much higher concentrations (approaching molar), as opposed to micromolar for the fatty acid effects.…”

Section: Fatty Acid Stimulation Of Activity and Restoration Ofmentioning

confidence: 99%

Fatty acids stimulate activity and restore respiratory control in a proton channel mutant of cytochrome c oxidase

Fetter¹,

Sharpe²,

Qian³

et al. 1996

FEBS Letters

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either an alanine (DI32A) or an asparagine (D132N) strongly diminishes oxygen reduction activity and abolishes proton pumping. D132 is a substantial distance from the oxygen-reactive site and mutation of the residue does not perturb the spectral properties of the metal centers [4]. Similar effects are seen when the corresponding residue in Escherichia coli cytochrome bo3 is mutated [5,6]).Another effect of the D132N/A mutations is that the reconstituted enzymes show anomalous responses to uncouplers and ionophores [4]. Valinomycin and CCCP, both singly and in combination, inhibit instead of stimulating turnover in reconstituted enzyme. The ionophores do not inhibit the purified enzyme directly and the oxygen reduction activity shows a similar decrease with increasing pH as wild-type, indicating that the inhibition is likely caused by changes in membrane potential (A~) and/or pH gradient (ApH) [4]. An understanding of these responses might clarify the pumping process itself. The present paper will show that (i) a normal membrane potential is formed by COV containing mutant enzyme and (ii) the defect of the D132A/N mutations can partially be overcome by addition of free fatty acids. The fatty acids also rectify the anomolous responses to uncouplers and ionophores, but repair of proton pumping has not been demonstrated. Materials and methods

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“…Crystals of both wild-type and the D96G mutant were trapped at a time in the photocycle that was subsequent to the release of a proton from the Schiff base, but before the uptake of a proton from the external aqueous medium. Under the conditions of our experiments, time-resolved visible spectroscopic experiments have shown that the M intermediate predominantly was accumulated in both wild-type bacteriorhodopsin (16) and the D96G mutant (17). Diffraction patterns recorded from the frozen crystals were processed to construct Fourier projection maps of the differences between the structures of the trapped intermediates and that of unilluminated bacteriorhodopsin.…”

mentioning

confidence: 99%

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

Subramaniam

Faruqi

Oesterhelt

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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We previously have presented evidence for prominent structural changes in helices F and G of bacteriorhodopsin during the photocycle. These changes were determined by carrying out electron diffraction analysis of illuminated two-dimensional crystals of wild-type bacteriorhodopsin or the Asp-96 3 Gly mutant that were trapped at a stage in the photocycle after light-driven proton release, but preceding proton uptake from the aqueous medium. Here, we report structural analysis of the long-lived O intermediate observed in the photocycle of the Leu-93 3 Ala mutant, which accumulates after the release and uptake of protons, but before the reisomerization of retinal to its initial all-trans state. Projection Fourier difference maps show that upon illumination of the Leu-93 3 Ala mutant, significant structural changes occur in the vicinity of helices C, B, and G, and to a lesser extent near helix F. Our results suggest that (i) all four helices that line the proton channel (B, C, F, and G) participate in structural changes during the late stages of the photocycle, and (ii) completion of the photocycle involves significant conformational changes in addition to those that are associated with steps in proton transport.Bacteriorhodopsin is a light-driven proton pump (1). Each cycle of proton transport is initiated by the all-trans to 13-cis photoisomerization of retinal and completed with the thermal re-isomerization of retinal and return of the protein to its initial conformation. A number of distinct intermediates that occur in the course of the photocycle have been identified by spectroscopic methods (2-4). Site-specific mutagenesis studies (5) and selection of transport-negative mutants (6) have identified key residues that play important roles in the formation and decay of these spectroscopic intermediates. Electron cryomicroscopic studies of two-dimensional crystals have resulted in the determination of a model for the structure of bacteriorhodopsin at atomic resolution (7,8), allowing a structural interpretation of the spectroscopic and biochemical experiments.To understand chemical aspects of the molecular mechanism of proton transport, it is also necessary to determine structural changes in the protein at different stages of the photocycle. A combination of neutron (9), x-ray (10-13), and electron diffraction (14, 15) experiments have begun to provide such information. Two general strategies have been used in these experiments. In one, structural changes have been observed by collecting diffraction data from wild-type bacteriorhodopsin where the photocycle has been slowed down by lowering the temperature, changing the pH, adding chaotropic reagents, or combining two or more of these variables. In the other strategy, mutants that have pronounced kinetic defects in specific stages of the photocycle have been used to trap and structurally characterize the corresponding intermediates. To a first approximation, the magnitude and nature of the structural changes determined by the different methods and by the diff...

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A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

Cited by 194 publications

References 38 publications

Leptosphaeria rhodopsin: Bacteriorhodopsin-like proton pump from a eukaryote

Leptosphaeria rhodopsin: Bacteriorhodopsin-like proton pump from a eukaryote

Fatty acids stimulate activity and restore respiratory control in a proton channel mutant of cytochrome c oxidase

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

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