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The active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pKa value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base proton in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pKa of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
April 1996,
Biochemistry,
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
May 1991,
Biochemistry,
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
September 2001,
Biochemistry,
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
July 1995,
Science (New York, N.Y.),
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
October 2001,
Journal of bioenergetics and biomembranes,
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
April 1992,
Journal of bioenergetics and biomembranes,
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
November 1985,
Seikagaku. The Journal of Japanese Biochemical Society,
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
February 2001,
Proceedings of the National Academy of Sciences of the United States of America,
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
January 1984,
Progress in clinical and biological research,
M Kataoka, and
H Kamikubo, and
F Tokunaga, and
L S Brown, and
Y Yamazaki, and
A Maeda, and
M Sheves, and
R Needleman, and
J K Lanyi
December 1997,
Biophysical journal,
