'American Society for Biochemistry & Molecular Biology (ASBMB)'
Abstract
With the aim of preparing a light-insensitive
bacteriorhodopsin-like pigment, bacterio-opsin expressed in
Escherichia coli was treated in phospholipid-detergent
micelles with the retinal analog II, in which the
C13-C14 trans-double bond cannot
isomerize due to inclusion in a cyclopentene ring. The
formation of a complex with a fine structure
(λmax, 439 nm) was first observed. This
partially converted over a period of 12 days to a
bacteriorhodopsin-like chromophore (ebR-II) with
λmax, 555 nm. An identical behavior has been
observed previously upon reconstitution of bleached purple
membrane with the analog II. Purification by gel filtration
gave pure ebR-II with λmax, 558 nm, similar
to that of light-adapted bacterio-opsin reconstituted with
all-trans retinal (ebR-I). Spectrophotometric titration of
ebR-II as a function of pH showed that the purple to blue
transition of bacteriorhodopsin at acidic pH was altered, and
the apparent pKa of Schiff base deprotonation at
alkaline pH was lowered by 2.4 units, relative to that of
ebR-I. ebR-II showed no light-dark adaptation, no proton
pumping, and no intermediates characteristic of the
bacteriorhodopsin photocycle. In addition, the rates of
reaction with hydroxylamine in the dark and in the light were
similar. These results show, as expected, that isomerization
of the C13-C14 double bond is required
for bacteriorhodopsin function and that prevention of this
isomerization confers light insensitivity