Effects of pH on rhodopsin photointermediates from lumirhodopsin to metarhodopsin II.
Time-resolved absorption difference spectra of membrane suspensions of bovine rhodopsin at pH 5, 6, 7, 8, 9, and 10 were collected in the time range from 1 micro s to 200 ms after laser photolysis with 7-ns pulses of 477-nm light. The data were analyzed using singular value decomposition (SVD) and global exponential fitting. At pH 7 the data agree well with previously obtained data (Thorgeirsson et al. (1993) Biochemistry 32, 13861-13872) with fits improved at all pH's by inclusion of a small component due to an absorbance change caused by rotational diffusion which is detectable even at magic angle polarization. A "square scheme" suggested to best explain the previous data, which involves two branches following decay of the lumi intermediate with pathways (1) lumi --> MI480 right harpoon over left harpoon MII and (2) lumi right harpoon over left harpoon MI380 --> MII, could be confirmed throughout the entire pH range. However, to account for the increased rate of the MII --> MI480 reaction in path 1 for rising pH values, we propose that the MII in the square scheme consists of deprotonated MII and protonated MIIH+ forms in rapid equilibrium with each other, resulting in an extended square scheme and increasing the number of 380-nm products from two to three. In addition to the kinetic processes described by the extended square scheme, above pH 8 fast ( approximately 10 micro s) and slow ( approximately 50 ms) components were found. The fast component was assigned to the decay of a blue-shifted lumi intermediate, and the slow component, resolvable only at pH 10, was assigned to formation of a 450 nm absorbing photoproduct.
Jäger
S
S
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
Szundi
I
I
Lewis
J W
JW
Mah
T L
TL
Kliger
D S
DS
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010782:Photolysis; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000378:metabolism; D013053:Spectrophotometry
C018586:lumirhodopsin; C013110:metarhodopsins; D012243:Rhodopsin