- مبلغ: ۸۶,۰۰۰ تومان
- مبلغ: ۹۱,۰۰۰ تومان
A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5 ms and 4.5 ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH 8.4, a spectrally silent electrogenic component with τ 0.25 ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH 5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer.
In this work we have resolved the kinetics of membrane potential generation, ΔΨ, coupled to the photocycle transitions in ESR, including those which involve its unusual proton donor (Lys96). Several differences were found in kinetics, amplitude and pH dependence of electrical components of ΔΨ in ESR with those in best studied H+ pump, BR. In ESR, the electrogenic events accompanying the M decay (reprotonation of the Schiff base) are primarily pH dependent, whereas in BR it is the N decay, which is pH dependent. This difference originates from different initial protonation state of the donor to the Schiff base and reaction of the photocycle in which proton is taken up (these are M to N1 in ESR and N1 to N2 in BR). Other differences are caused by different mechanism of proton release in ESR and BR and counterion structure (presence of ionizable His57 close to Asp85 in ESR). Formation of the M intermediate (upon Schiff base deprotonation) is associated with two positive electrogenic phases (~3 μs and ~40 μs) of ΔΨ generation by ESR, which comprise ~9% of the total photoelectric response and correspond to the distance between the Schiff base and the primary proton acceptor Asp85. This is about 3 times less than the contribution of the 50 μs component in BR, which includes also movement of Arg82 to the proton release group and release of a proton to the bulk. The subsequent electrogenic reprotonation of the Schiff base of ESR from the cytoplasmic side (M ↔ N1 ↔ N2/O transitions) occurs with τ ~ 0.5 ms and 4.5 ms. At pH 8,4, the fast component ceases and the Schiff base reprotonation slows down, indicating that capturing a proton from the bulk becomes the rate limiting step.