Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin.

Abstract

Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of HI, 1H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor ru-st reprotonates the Schiffbase in the decay of M to N and is then reprotonated from the cytoplasm in the N -O 0 transition. The decay rate of N is proportional to [HI].By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (7)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like HI, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become >100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiffbase in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium halobium that transports H+ ions from the cytoplasm to the extracellular space with a stoichiometry of one proton per cycle (1). The transmembrane H+ translocation involves distinct electrogenic steps associated with H+ ejection from the protein interior into the periplasm and the subsequent H+ rebinding from the cytoplasmic side of the membrane. The proton uptake occurs on the millisecond time scale and is apparently coupled to the reprotonation of the Schiff base (SB) and the decay of the M intermediate of the photochemical cycle. Fourier transform infrared (FTIR) spectroscopy first indicated that several aspartate carboxyl groups undergo protonation changes during the photocycle (2, 3). Site-directed mutagenesis of bR showed that substitution of aspartate residues at positions 85, 96, and 212 by asparagine reduced the proton pumping activity to a few percent (4) and revealed, in combination with FTIR measurements, the protonation states of specific aspartate residues in various photocycle intermediates (5, 6). In the mutant D96N, the kinetics of proton uptake is affected (7-9). We recently showed that the low steady-state proton pumping act...

High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin

“…1d). UV-visible spectroscopy 26 has shown that the lifetime of the M-intermediate of D96N and WT is prolonged at alkaline pH; in concordance with this fact is the observation here with D96N that the decay detected by high-speed AFM showed a strong pH dependence (inset, Fig. 1d).…”

“…We used the D96N bR mutant, which has a longer photocycle (~10 s) than that of WT (~10 ms) but retains proton pumping ability 26 . Figure 1a We analyzed the "mass center" positions of the individual monomers imaged during the dark-illumination cycles.…”

Fatty acids stimulate activity and restore respiratory control in a proton channel mutant of cytochrome c oxidase

“…If the fatty acid is complementing the loss of the D132 carboxyl, or interacting at another site to improve proton access, this resembles the chemical rescue phenomenon observe with carboxylate mutants of other proton transferring membrane proteins. In mutated bacteriorhodopsin [14,15] and bacterial photosynthetic reaction center [16] activity can be restored by weak acid anions such as azide. These water soluble acids are required at much higher concentrations (approaching molar), as opposed to micromolar for the fatty acid effects.…”

Time‐resolved surface charge change on the cytoplasmic side of bacteriorhodopsin

“…The 1.5 ms component in the M-decay of V101C-AF is most likely due to the M-to-N/O transition, in which the Schiff base is reprotonated from aspartate-96 [2,3]. The 63.9 ms component is probably due to the decay of N and O which are coupled to the M-decay by back reactions [16]. This is supported by photocycle measurements at 650 nm (data not shown), which indicate that O returns to the initial state with a decay time of 61.7 ms.…”