Temperature dependence of the lumirhodopsin I-lumirhodopsin II equilibrium.
Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were made at delays from 1 micros to 2 ms after photoexcitation of bovine rhodopsin in hypotonically washed membrane suspensions over a range of temperature from 10 to 35 degrees C. The purpose was to better understand the reversibility of the Lumi I-Lumi II process that immediately precedes Schiff base deprotonation in the activation of rhodopsin under physiological conditions. To prevent artifacts due to rotation of rhodopsin and its photoproducts in the membrane, probe light in the time-resolved absorbance studies was polarized at the magic angle (54.7 degrees) relative to the excitation laser polarization axis. The difference spectrum associated with the Lumi I to Lumi II reaction was found to have larger amplitude at 10 degrees C compared to higher temperatures, suggesting that a significant back-reaction exists for this process and that an equilibrated mixture forms. The equilibrium favors Lumi I entropically, and van't Hoff plot curvature shows the reaction enthalpy depends on temperature. The results suggest that Lumi II changes its interaction with the membrane in a temperature-dependent way, possibly binding a membrane lipid more strongly at lower temperatures (compared to its precursor). To elucidate the origin of the time-resolved absorbance changes, linear dichroism measurements were also made at 20 degrees C. The time constant for protein rotation in the membrane was found to be identical to the time constant for the Lumi I-Lumi II process, which is consistent with a common microscopic origin. We conclude that Lumi II (the last protonated Schiff base photointermediate under physiological conditions) is the first photointermediate whose properties depend on the protein-lipid environment.
Szundi
Istvan
I
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Epps
Jacqueline
J
Lewis
James W
JW
Kliger
David S
DS
D000818:Animals; D002417:Cattle; D008027:Light; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism*; D012545:Schiff Bases; D013696:Temperature; D013816:Thermodynamics
D012545:Schiff Bases; C018586:lumirhodopsin; D012243:Rhodopsin