INTERACTION OF PROTON AND CHLORIDE TRANSFER PATHWAYS IN RECOMBINANT BACTERIORHODOPSIN WITH CHLORIDE TRANSPORT ACTIVITY - IMPLICATIONS FOR THE CHLORIDE TRANSLOCATION MECHANISM

Citation
Ls. Brown et al., INTERACTION OF PROTON AND CHLORIDE TRANSFER PATHWAYS IN RECOMBINANT BACTERIORHODOPSIN WITH CHLORIDE TRANSPORT ACTIVITY - IMPLICATIONS FOR THE CHLORIDE TRANSLOCATION MECHANISM, Biochemistry, 35(50), 1996, pp. 16048-16054
Citations number
35
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
35
Issue
50
Year of publication
1996
Pages
16048 - 16054
Database
ISI
SICI code
0006-2960(1996)35:50<16048:IOPACT>2.0.ZU;2-X
Abstract
When the protonated retinal Schiff base dissociates in the photocycle of the proton pump bacteriorhodopsin, asp-85 is the proton acceptor. R eplacing this residue with threonine confers halorhodopsin-like proper ties on the protein, including chloride transport [Sasaki, J., Brown, L, S., Chon, Y.-S., Kandori, H., Maeda, A., Needleman, R., & Lanyi, J. K. (1995) Science 269, 73-75], However, the electrostatic interaction between the vicinity of residue 85 and glu-204, a residue located abo ut 10 Angstrom away near the extracellular surface, that is a part of the proton transport mechanism, should still exist, We find that in th e D85T mutant glu-204 becomes protonated when chloride is added, This indicates that the binding of chloride at thr-85 must be equivalent to deprotonation of asp-85. The protonation state of glu-204 reports the refore on the presence or absence of chloride bound at thr-85. During the chloride-transport cycle of D85T, but not D85T/E204Q, fluorescein and pyranine detect the transient release of protons from the protein to the surface and the bulk. The release and the subsequent uptake of the protons occur during the rise and decay of a red-shifted photointe rmediate, respectively, and confirm the earlier suggestion that this s tate has the same role in the chloride transport as the hi intermediat e in the proton transport. Consistent with the red-shift of the absorp tion maximum, the chloride bound near the Schiff base had already move d away, presumably to be released at the cytoplasmic surface, but anot her chloride ion has not yet been taken up from the extracellular surf ace. The switch of the connectivity of the chloride binding site from the cytoplasmic to the extracellular membrane surface must occur there fore during the lifetime of this photointermediate.