The structure and function of a rhodopsin-like pigment bacteriorhodopsin, discovered and isolated from the membranes of the halophile Halobacterium halobium, were studied. Intermediatesm appearing in the cyclic photoreaction that drives the proton translocation, were spectroscopically characterized. The charge translocation in membrane monolayers and mulitlayers placed between electrodes were kinetically resolved. A model was developed for the proton translocation process, in which the isomerization of the retinal Schiff base decreases its pK to drive the proton off and simultaneaouly changes the connectivity from the cytoplasmic surface to the external surface. The stoichiometry of proton pumping in intact cells and the effect of the light generated electrochemical potential on the kinetics of the photoreaction cycle and the synthesis of ATP were investigated

Bogomolni, R. A.

Stoeckenius, W.

English

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PHOTOENERGY TRANSDUCTION IN HALOBACTERIUM HALOBIUM
FINAL TECHNICAL REPORT
Principal Investigators:
Walther Stoeckenius and Roberto A. Bogomolni
July 1, 1975 through October 31, 1985
The Regents of The Universitiy of California
Contracts and Grants Office
145 Irving Street
San Francisco, CA 94143
Grant Number NSG-7151
^{NASA-CB-176673) PH01CENEEGJ 1BANSDUCTION N86-25970
IN H A L O B A C T E B I U M H A L O E I O M Final Beport, 1
Jul. ,1975 - 31 Oct. 1865 (California Univ.)
9 p HC A02/HF A01 CSCL 06C Dnclas
G3/51
https://ntrs.nasa.gov/search.jsp?R=19860016498 2020-03-20T14:24:38+00:00Z
FINAL TECHNICAL REPORT
Summary of Project. This grant was awarded to study the structure and function
•-*:
of a rhodopsin-like pigment bacteriorhodopsin (bR) / which we had discovered in cell
membranes of the extreme nalophile Halobacterium halobium and isolated in pure form.
Its function was shown to be that of a light-energy transducer, which generates an
electrochemical potential across the cell membrane/ by translocating protons from the
cytoplasm to the outside medium. The cell then uses this gradient to drive other
metabolic processes and locomotion.
Under this grant we spectroscopically characterized intermediates which appear
in the cyclic photoreaction that drives the proton translocation. For this we esta-
blished time-resolved absorption spectroscopy which covered the time-range from 10
to 10 s. For higher time resolution and resonance Raman experiments we collaborated
with other groups. Wa established that the Schiff base which links the chromophore
retinal to the protein undergoes a transient deprotonation during the photocycle, and
also an all-trans to 13-cis isomerization. We further showed that after deprotona-
tion of the Schiff base/ a proton is ejected from the external side and later one
proton taken up on the cytoplasmic side of the transmembrane protein, bR. We have
also kinetically resolved the charge translocation in membrane monolayers and mul-
tilayers placed between electrodes. We have determined the angle between the chromo-
phore transition movement and the plane of the membrane and shown that there is lit-
tle if any change during the photoreaction cycle.
Bacteriorhodopsin occurs in the form of a crystalline two-dimensional lattice in
the plasma membrane, which allows determination of its structure with near atomic
resolution. Apparently, its polypeptide chain crosses the membrane seven times in
ex-helical configuration. We have used reconstitution of bR from the opsin and
NSG-7151 2 W. Stoeckenius
retinal with one or the other component deuterated to obtain localization of retinal
in the structure by neutron diffraction. It showed the p -ionone ring in the center
of the membrane profile. The in-plane localization was ambiguous and has recently
been superceded as a result of similar experiments carried out with the far superior
neutron source available at Grenoble. In collaboration with R. Stroud, we have
determined that an earlier determination of the binding site in the amino acid
sequence of bR was erroneous and that the G-amino group of Lys 216 is the correct
site. This collaboration also led to a higher resolution structure which limited the
possible assignments of amino acid sequence to the rod-like structures seen in the
structural model.
Based on these data/ we have developed a model for the proton translocation pro-
cess/ in which the isomerization of the retinal Schiff base decreases its pK to drive
the proton off and simultaneously changes the connectivity from the cytoplasmic sur-
face to the external surface. Proton conduction is assumed to occur via transmem-
brane chains of hydrogen-bonded groups. We have tried to follow the change in acces-
sability of the Schiff base using nanosecond electron beam pulses to rapidly reduce
the Schiff base and stably link it to the protein. We further hoped to disprove con-
clusively a postulated transient change in the Schiff base linkage from Lys 41 to 216
in the photocycle. These experiments/ however/ proved so time-consuming and costly
that they did not progress beyond a feasibility study.
We have further investigated the stoichiometry of proton pumping in intact cells
and the effect of the light-generated electrochemical potential on the kinetics of
the photoreaction cycle and the synthesis of ATP. The latter experiments yielded
strong evidence that in addition to a classical chemi-osmotic mechanism/ a more
direct energy-coupling must exist.
NSG-7151 3 W. Stoeckenius
Finally, we have used the techniques developed for the study of ]1. halobium to
investigate the occurence of bacteriorhodopsin and similar pigments in isolates from
several natural sources/ mainly the southern Sinai coast, the viestern Sahara and Baja
California. Photoactive pigments similar or identical to bR or-the closely related
light-driven chloride pump halorhodopsin (hR) were found in many. For some, we have
also demonstrated the corresponding light-driven ion movements and ATP-synthesis. In
addition, similar pigments with a much slower photocycle were observed, which are
apparently similar to the recently discovered sensory bacterial rhodopsins, which
mediate phototactic responses.
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Photoenergy transduction in halobacterium halobium

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