Halide Dependence of the Halorhodopsin Photocycle as Measured by Time-Resolved Infrared Spectra

Abstract

Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3 micros to 100 ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of tau(1) approximately 150 micros and tau(2) approximately 16 ms in the presence of either halide, with tau(2) describing the return to the starting (hR) state. Comparison to previous low-temperature FTIR spectra of hR intermediates confirms that characteristic hK and hL spectral features are both present before the tau(1) decay, in a state previously defined as hK(L) (Dioumaev, A., and M. Braiman. 1997. Photochem. Photobiol. 66:755-763). However, the relative sizes of these features depend on which halide is present. In Br-, the hL features are clearly more dominant than in Cl-. Therefore, the state present before tau(1) is probably best described as an hK(L)/hL(1) equilibrium, instead of a single hK(L) state. Different halides affect the relative amounts of hK(L) and hL(1) present, i.e., Cl- produces a much more significant back-reaction from hL(1) to hK(L) than does Br-. The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump.

The Protonated Schiff Base of Halorhodopsin from Natronobacterium pharaonis is Hydrolyzed at Elevated Temperatures†

“…Three chloride binding sites were proposed in which one is the transport site which has affinity of 2.5 mM located in the vicinity of the PSB. This location was defined by X-ray studies and suggested previously by FTIR and resonance Raman data (38,39) and absorption spectroscopy (30,40). The second binding site is the cytoplasmic release site with an affinity of 5.7 M, and a third one was suggested on the extracellular site (41) with a binding constant of 200 mM.…”

A Vibrational Spectral Maker for Probing the Hydrogen-Bonding Status of Protonated Asp and Glu Residues

“…3 B shows The color codes, the methods for frequency and energy calculations, and the definition of hydrogen-bond lengths are the same as those in Table 4. 1.80 Å 1742 [8] Bacteriorhodopsin D96 HOÿC¼O 2.79 1MOM [9] 1.43 Å 1737 [10] PYP E46 HOÿC¼O 2.58 1NWZ [11] 0.82 Å 1735 [12] Rhodopsin E134 HOÿC¼O 2.53/2.51 y 1L9H [2] 2.60 Å 1735 [13] Reaction center E104 HOÿC¼O 2.78 1M3X [14] 2.55 Å 1734 [8] Bacteriorhodopsin D115 HOÿC¼O 2.88 1MOM [9] 1.43 Å 1734 [1] Rhodopsin E122 (Fahmy et al, 1993); [2] (Okada et al, 2002); [3] (Schmidt et al, 2004); [4] (Tsukihara et al, 2003); [5] (Svensson-Ek et al, 2002); [6] (Hutson et al, 2001); [7] (Kolbe et al, 2000); [8] (Sasaki et al, 1994); [9] (Lanyi and Schobert, 2002); [10] (Xie et al, 1996); [11] ; [12] (DeLange et al, 1999); [13] (Breton et al, 1997); [14] (Camara-Artigas et al, 2002); [15] (Koutsoupakis et al, 2004); [16] (Soulimane et al, 2000). y These are the two hydrogen-bond lengths corresponding to two structures of the same protein in the same state from one PDB data.…”

Halide Transport through Biological Membranes

“…This chromophore causes the purple-like color of the unphotolyzed halorhodopsin state with an absorbance maximum λ max of 578 nm. [55,56] Because of the long distance and the relative geometrical arrangement, a strong hydrogen bond between the chloride and the Schiff base nitrogen is not expected in the halorhodopsin state. The 23 kcal/mol of remaining photon energy are mostly stored in the K-state by a twisted conformation of the retinal chromophore and drive the ongoing conformational changes of the protein which accompany unidirectional ion transport.…”

“…[55] Alternative or in addition to the piston model, where movements of the T111 residue drive the ion translocation, some form of "iondragging" was considered before where the halide ion follows the flipped dipole of the N-H Schiff base bond and keeps a strong hydrogen bond with the protonated Schiff base. [55] Alternative or in addition to the piston model, where movements of the T111 residue drive the ion translocation, some form of "iondragging" was considered before where the halide ion follows the flipped dipole of the N-H Schiff base bond and keeps a strong hydrogen bond with the protonated Schiff base.…”