Properties of Asp212—-Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 micros to 75 micros with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.

Section: A Model For Describing the Ph Dependence Of The Proton Affinity Of Asp 85 : Implications For The Ph Dependencies Of Dark Adaptatsupporting

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Two Groups Control Light-Induced Schiff Base Deprotonation and the Proton Affinity of Asp85 in the Arg82His Mutant of Bacteriorhodopsin

Imasheva

Balashov

Ebrey

et al. 1999

“…The mutant strains of H. salinarum, A53V, D85N, and E204Q were provided by Prof. R. Needleman of Wayne State University and Prof. J. K. Lanyi of University of California, Irvine. The strains, A51G, A81G, A84G, A184G, P186A, and A215G were constructed as described by Needleman et al (1991). H. salinarum strain S9 and the mutant strains were grown in the temporary synthetic (TS) medium (Onishi et al, 1965), in which unlabeled L-Ala was replaced by [3-13 C]Ala.…”

Section: Methodsmentioning

confidence: 99%

Regio-selective Detection of Dynamic Structure of Transmembrane α-Helices as Revealed from 13C NMR Spectra of [3-13C]Ala-labeled Bacteriorhodopsin in the Presence of Mn2+ Ion

Tuzi

Hasegawa

Kawaminami

et al. 2001

13 C Nuclear magnetic resonance (NMR) spectra of [3- 13 C]Ala-labeled bacteriorhodopsin (bR) were edited to give rise to regio-selective signals from hydrophobic transmembrane ␣-helices by using NMR relaxation reagent, Mn 2ϩ ion. As a result of selective suppression of 13 C NMR signals from the surfaces in the presence of Mn 2ϩ ions, several 13 C NMR signals of Ala residues in the transmembrane ␣-helices were identified on the basis of site-directed mutagenesis without overlaps from 13 C NMR signals of residues located near the bilayer surfaces. The upper bound of the interatomic distances between 13 C nucleus in bR and Mn 2ϩ ions bound to the hydrophilic surface to cause suppressed peaks by the presence of Mn 2ϩ ion was estimated as 8.7 Å to result in the signal broadening to 100 Hz and consistent with the data based on experimental finding. The Ala C ␤ 13 C NMR peaks corresponding to Ala-51, Ala-53, Ala-81, Ala-84, and Ala-215 located around the extracellular half of the proton channel and Ala-184 located at the kink in the helix F were successfully identified on the basis of 13 C NMR spectra of bR in the presence of Mn 2ϩ ion and site-directed replacement of Ala by Gly or Val. Utilizing these peaks as probes to observe local structure in the transmembrane ␣-helices, dynamic conformation of the extracellular half of bR at ambient temperature was examined, and the local structures of Ala-215 and 184 were compared with those elucidated at low temperature. Conformational changes in the transmembrane ␣-helices induced in D85N and E204Q and its long-range transmission from the proton release site to the site around the Schiff base in E204Q were also examined.

“…A considerable similarity in the three-dimensional structures was found between HR and BR (Havelka et al, 1995;Kolbe et al, 2000). Several highly conserved amino acids in the binding pocket of retinal, such as Asp-212 in BR (residue 238 in sHR and residue 252 in pHR) and Arg-82 (residue 108 in sHR and residue 123 in pHR), take part in a counterion complex which stabilizes the protonated Schiff base (Needleman et al, 1991;Balashov et al, 1992;Cao et al, 1993).…”

Section: Introductionmentioning

confidence: 98%

The Nitrate Transporting Photochemical Reaction Cycle of the Pharaonis Halorhodopsin

Bálint

Lakatos

Ganea

et al. 2004

Time-resolved spectroscopy, absorption kinetic and electric signal measurement techniques were used to study the nitrate transporting photocycle of the pharaonis halorhodopsin. The spectral titration reveals two nitrate-binding constants, assigned to two independent binding sites. The high-affinity binding site (K(a) = 11 mM) contributes to the appearance of the nitrate transporting photocycle, whereas the low-affinity constant (having a K(a) of approximately 7 M) slows the last decay process in the photocycle. Although the spectra of the intermediates are not the same as those found in the chloride transporting photocycle, the sequence of the intermediates and the energy diagrams are similar. The differences in spectra and energy levels can be attributed to the difference in the size of the transported chloride or nitrate. Electric signal measurements show that a charge is transferred across the membrane during the photocycle, as expected. A new observation is an apparent release and rebinding of a small fraction of the retinal, inside the retinal pocket, during the photocycle. The release occurs during the N-to-O transition, whereas the rebinding happens in several seconds, well after the other steps of the photocycle are over.