Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate

Abstract

The kinetics of the photoreversal reaction of the M-intermediate of bacteriorhodopsin (bR) was investigated by time-resolved optical absorption spectroscopy and photovoltage measurements using double-flash excitation (a green flash (532 nm) followed by a blue flash (400 nm) after a variable delay). The sign of the photovoltage and the 1H/2H kinetic isotope effect indicate that the Schiff base is reprotonated by a group between the Schiff base and the extracellular surface, probably Asp85. Analysis of the kinetic data shows that the charge movement in 150 mM KCl at 12 degrees C is characterized by two components with time constants of approximately 100 ns and approximately 600 ns, respectively, which are independent of the delay time between the flashes and the pH. The amplitudes of the fast and slow components depend on the delay and the pH. The slower component starts to contribute to the charge movement only after delays longer than 100 micros, is absent at low pH, and increases in amplitude with a pKa of approximately 6. Because the proton release group deprotonates after 70-100 micros and has a transient pKa of 5.8, these results suggest the following assignment: the fast and the combination of fast and slow components represent photoreversal from two M states, with the release group protonated and deprotonated, respectively. The slow phase of the photoreversal starts from a state with the release group deprotonated, and with the pK of Asp85 elevated, and is probably due to the restoration of the pK of Asp85 to its initial low value. This provides further evidence for coupling between the pK's of Asp85 and the release group and suggests that proton release is the first step in the reprotonation switch. At alkaline pH the amplitude of the electrical signal from the back photoreaction decreases with an apparent pK of 8, without a corresponding decrease in the amount of M. At neutral pH the movement of the positively charged guanidinium group of Arg82 from a position near the release group on the surface to Asp85 makes a substantial contribution to the electrical photoreversal amplitude. Above the pK of the release group in the unphotolysed state (approximately 8), Arg82 stays near the surface, leading to a corresponding signal reduction.

Early and Late M Intermediates in the Bacteriorhodopsin Photocycle: A Solid-State NMR Study

“…Our observation of the two subtly different substates of M o may be related to the two subtly different M's detected in studies of the photo-induced back-reaction (29)(30)(31), and thus to proton release from the membrane (30). This possibility could be tested by further NMR studies monitoring the pH dependence of the ratios of the two M o substates down to the pHs at which the slow component in the M back-photoreaction disappears, putatively because proton release is delayed until later in the photocycle.…”

Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

“…A conformational change in helix G introduced by the mutation G231C can lead to a different interaction of the Asp-212 side chain with the neighboring residues in R82A/G231C in such a way that the side chain of Asp-212 no longer participates in the complex counterion. Recent time-resolved electrical measurements (19) provide evidence that the movement of the positively charged arginine 82 side chain makes a substantial contribution to the amplitude of the electrical signal in wt. This amplitude is clearly reduced in the mutants G231C and R82A/ G231C.…”

“…The proposed movement of this side chain toward the extracellular surface upon formation of the M-intermediate, based on computer calculations (18), is not yet established. However, recent time-resolved electrical measurements support this proposal (19).…”

Progress toward an explicit mechanistic model for the light‐driven pump, bacteriorhodopsin

“…Protonation of Asp‐85 is linked to the release of a proton to the extracellular surface [15,16], and at pH well above the p K for the release (p K =5–6), the protonation equilibrium will be shifted far toward full deprotonation of the Schiff base. Thus, the proton release shifts the next step, the M 1 to M 2 transition, toward M 2 [24–26]. At pH<5, proton release does not occur at this time, and the L state persists along with M 1 and M 2 during the further progress of the photocycle [24,27,28].…”

“…The access change in the pump is the second question of importance. Kinetically, the protonation switch, from the extracellular to the cytoplasmic side, is represented by the M 1 →M 2 step, which is a unidirectional reaction under some conditions but an equilibration under others [24–26,28]. In principle, the switch may be a combination of changes of the p K s of the proton acceptor and donor, and the making and breaking of proton conductive pathways to the extracellular and cytoplasmic directions.…”