title: Proton uptake and release are rate-limiting steps in the photocycle of the bacteriorhodopsin mutant E204Q.
abstract: 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.
authors.affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana 61801, USA.
authors.forename: S
authors.initials: S
authors.lastname: Misra
authors.forename: R
authors.initials: R
authors.lastname: Govindjee
authors.forename: T G
authors.initials: TG
authors.lastname: Ebrey
authors.forename: N
authors.initials: N
authors.lastname: Chen
authors.forename: J X
authors.initials: JX
authors.lastname: Ma
authors.forename: R K
authors.initials: RK
authors.lastname: Crouch
mesh_terms: 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
chemical_list: D001386:Azides; D011522:Protons; D001436:Bacteriorhodopsins; D017666:Deuterium Oxide