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