Proton movement and photointermediate kinetics in rhodopsin mutants.
The role of ionizable amino acid side chains in the bovine rhodopsin activation mechanism was studied in mutants E134Q, E134R/R135E, H211F, and E122Q. All mutants exhibited bathorhodopsin stability on the 30 ns to 1 micros time scale similar to that of the wild type. Lumirhodopsin decay was also similar to that of the wild type except for the H211F mutant where early decay (20 micros) to a second form of lumirhodopsin was seen, followed by formation of an extremely long-lived Meta I(480) product (34 ms), an intermediate which forms to a much reduced extent, if at all, in dodecyl maltoside suspensions of wild-type rhodopsin. A smaller amount of a similar long-lived Meta I(480) product was seen after photolysis of E122Q, but E134Q and E134R/R135Q displayed kinetics much more similar to those of the wild type under these conditions (i.e., no Meta I(480) product). These results support the idea that specific interaction of His211 and Glu122 plays a significant role in deprotonation of the retinylidene Schiff base and receptor activation. Proton uptake measurements using bromcresol purple showed that E122Q was qualitatively similar to wild-type rhodopsin, with at least one proton being released during lumirhodopsin decay per Meta I(380) intermediate formed, followed by uptake of at least two protons per rhodopsin bleached on a time scale of tens of milliseconds. Different results were obtained for H211F, E134Q, and E134R/R135E, which all released approximately two protons per rhodopsin bleached. These results show that several ionizable groups besides the Schiff base imine are affected by the structural changes involved in rhodopsin activation. At least two proton uptake groups and probably at least one proton release group in addition to the Schiff base are present in rhodopsin.
Lewis
James W
JW
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Szundi
Istvan
I
Kazmi
Manija A
MA
Sakmar
Thomas P
TP
Kliger
David S
DS
D000818:Animals; D002417:Cattle; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics*; D012545:Schiff Bases / Q000737:chemistry; D013057:Spectrum Analysis
D011522:Protons; D012545:Schiff Bases; D012243:Rhodopsin