Proton uptake and release are rate-limiting steps in the photocycle of the bacteriorhodopsin mutant E204Q.
In the absence of the putative proton release group, E204, the second half of the photocycle of the E204Q mutant of bacteriorhodopsin is slowed down more than 10-fold compared to the wild type. The effects of pH and D2O on the M decay and O formation rates in E204Q suggest that proton uptake occurs concurrently with the N <--> O transition, possibly coupled with the thermal reisomerization of the retinal. Hence, one of the rate-limiting steps in the slow E204Q photocycle is proton uptake from the outside medium, coincident with the decay of the slow component of M (the N <--> O transition). The second rate-limiting step is the long lifetime of decay of the O state, due to a high activation barrier for the deprotonation of D85 in the O --> bR step of the E204Q photocycle. Addition of the weakly acidic anions azide, cyanate, or formate accelerates the decay of the O intermediate, and restores the total photocycling time to that observed in the wild-type pigment, by accelerating the deprotonation of D85. We also find that azide similarly accelerates the decay of O in the wild type under conditions in which E204 does not deprotonate during the photocycle (pH < 6). It has previously been shown that azide and other weak acids can influence proton transfers in the cytoplasmic half of the protein [Tittor, J., Soell, C., Oesterhelt, D., Butt, H.-J., & Bamberg, E. (1989) EMBO J. 8, 3477-3482]; we suggest that these weak acids can affect proton transfers in the extracellular half of the protein as well.
Misra
S
S
Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana 61801, USA.
Govindjee
R
R
Ebrey
T G
TG
Chen
N
N
Ma
J X
JX
Crouch
R K
RK
D001386:Azides / Q000494:pharmacology; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017666:Deuterium Oxide / Q000378:metabolism; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011522:Protons; D018717:Purple Membrane / Q000378:metabolism; D013696:Temperature
D001386:Azides; D011522:Protons; D001436:Bacteriorhodopsins; D017666:Deuterium Oxide