Resonance Raman study on binding of chloride to the chromophore of halorhodopsin

Abstract

The resonance Raman spectrum of halorhodopsin, a retinoid protein with light-dependent chloride pumping activity, was observed in the presence and absence of chloride in H20 and D20. The frequency of the in-phase C=C stretching mode was shifted from 1528 to 1530 cm-' upon removal of chloride, in accordance with what was expected from the shift of the wavelength for the absorption maximum from 576 to 567 nm. The chloride effect was also found with the C=NH stretching modes of the protonated Schiff base, which were located at 1635 and 1642 cm-' in the presence and absence of chloride, respectively. However, the corresponding frequencies of the deuterated Schiff base linkage exhibited little dependence on chloride. The N-H bending mode of the protonated Schiff base was assigned to the Raman line at 1352 cm-' since it was apparently replaced by the Raman line at 977 cm-' upon deuteration. This line did not undergo any chloride effect. The Raman spectrum in the 1150-1250-cm-' region was unaffected by chloride and was similar to that of all-trans-bacteriorhodopsin. These observations and simple vibrational calculations suggest that chloride is not hydrogen bonded to the Schiff base proton in a trivial manner but that binding of chloride changes a limited number of force constants of the C=NH moiety, presumably through a direct interaction with the ?r orbital of the Schiff base or indirectly through a local structural change of the protein.

Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein 1 1Edited by A.R.Fersht

“…In HR from H. salinarum the exclusion size of anions was determined at an ionic radius of 1.65 A Ê , i.e. sulfate does not cause color changes in HR (Maeda et al, 1985;Schobert & Lanyi, 1986). In contrast, a small but measurable blue shift is demonstrated for sulfate in D85T.…”

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

“…One of the sites (Site I) is relatively non-specific for C1-and will bind many other monovalent anions ). This site is near, but not on, the retinal Schiff base chromophore (Steiner et al 1984, Maeda et al 1985. Chloride binding to this site raises the pKa of the Schiff base by about 2 units (Steiner et al 1984) and stabilizes the 580 nm intermediate in the photocycle (Steiner et al 1984, Lanyi andVodyanoy 1986).…”

Cation Binding to Halorhodopsin

“…17−20 These have shown that Cl − ions usually bind to the vicinity of the protonated Schiff base linkage, as studied by Raman spectroscopy. 21,22 The high-resolution structure of shR reveals that the chloride anion forms part of the retinal protonated Schiff base (PSB) counterion complex and replaces Asp85 of BR. In the case of halorhodopsin from N. pharaonis, Sato et al proposed the existence of Cl − binding or interacting sites in both the extracellular and cytoplasmic channels.…”