Two substates in the O intermediate of the light-driven proton pump archaerhodopsin-2

The proton pumping cycle of archaerhodopsin-2 (aR2) was investigated over a wide pH range and at different salt concentrations. We have found that two substates, which are spectroscopically and kinetically distinguishable, occur in the O intermediate. The first O-intermediate (O1) absorbs maximumly at ~580 nm, whereas the late O-intermediate (O2) absorbs maximumly at 605 nm. At neutral pH, O1 is in rapid equilibrium with the N intermediate. When the medium pH is increased, O1 becomes less stable than N and, in proportion to the amount of O1 in the dynamic equilibrium between N and O1, the formation rate of O2 decreases. By contrast, the decay rate of O2 increases ~100 folds when the pH of a low-salt membrane suspension is increased from 5.5 to 7.5 or when the salt concentration is increased to 2 M KCl. Together with our recent study on two substates in the O intermediate of bacteriorhodopsin (bR), the present study suggests that the thermally activated re-isomerization of the retinylidene chromophore into the initial all-trans configuration takes place in the O1-to-O2 transition; that is, O1 contains a distorted 13-cis chromophore. It is also found that the pKa value of the key ionizable residue (Asp101^aR2, Asp96^bR) in the proton uptake channel is elevated in the O1 state of aR2 as compared to the O1 state of bR. This implies that the structural property of O1 in the aR2 photocycle can be investigated over a wide pH range.