pH dependence of light-driven proton pumping by an archaerhodopsin from Tibet: comparison with bacteriorhodopsin

Abstract

The pH-dependence of photocycle of archaerhodopsin 4 (AR4) was examined, and the underlying proton pumping mechanism investigated. AR4 is a retinal-containing membrane protein isolated from a strain of halobacteria from a Tibetan salt lake. It acts as a light-driven proton pump like bacteriorhodopsin (BR). However, AR4 exhibits an "abnormal" feature--the time sequence of proton release and uptake is reversed at neutral pH. We show here that the temporal sequence of AR4 reversed to "normal"--proton release preceding proton uptake--when the pH is increased above 8.6. We estimated the pK(a) of the proton release complex (PRC) in the M-intermediate to be approximately 8.4, much higher than 5.7 of wide-type BR. The pH-dependence of the rate constant of M-formation shows that the pK(a) of PRC in the initial state of AR4 is approximately 10.4, whereas it is 9.7 in BR. Thus in AR4, the chromophore photoisomerization and subsequent proton transport from the Schiff base to Asp-85 is coupled to a decrease in the pK(a) of PRC from 10.4 to 8.4, which is 2 pK units less than in BR (4 units). This weakened coupling accounts for the lack of early proton release at neutral pH and the reversed time sequence of proton release and uptake in AR4. Nevertheless the PRC in AR4 effectively facilitates deprotonation of primary proton acceptor and recovery of initial state at neutral pH. We found also that all pK(a)s of the key amino acid residues in AR4 were elevated compared to those of BR.

FIGURE 1

(A) Flash-induced absorption changes of the pH-sensitive dye pyranine in suspensions of BR or AR4, detected at 456 nm. The absorption changes obtained as a difference between traces taken after and before addition of pyranine. ΔOD₄₅₆ are normalized to OD₄₁₂,_max, the amount of flash-induced M-intermediate formed in the photocycle of each protein. The downward curve corresponds to acidification of the medium (proton release first), whereas the upward curve corresponds to proton uptake. Samples were measured in 150 mM KCl aqueous solution containing 25% glycerol. (B) pH-dependence of flash-induced absorption changes of pyranine in AR4 suspension. The sample was measured in 100 mM NaCl and 20 mM KCl.

FIGURE 2

pH-dependence of flash-induced absorption changes of thymol blue in suspensions of AR4 at different pHs, detected at 456 nm. The absorption changes were obtained as a difference between traces taken after and before addition of thymol blue. The increase of absorption corresponds to acidification of the medium (proton release), whereas the decrease corresponds to proton uptake. The sample was measured in 100 mM NaCl and 20 mM KCl. The molar concentration of thymol blue is 0.03 mM, 2–3 times of that of AR4.

FIGURE 3

(A) Absorption spectra of light-adapted AR4. (B) Wavelength dependence of the maximum light-induced absorption changes of AR4, measured every 10 nm from 350 to 720 nm (pH 6.8, 75 mM K₂SO₄, 20°C).

FIGURE 4

(A) Absorption spectra of dark-adapted claret membranes (75 mM K₂SO₄, 20°C). Spectra 1–11 were measured at pH 6.90, 6.10, 5.45, 5.00, 4.65, 4.25, 3.75, 3.40, 3.05, 2.50, and 2.05, respectively. (B) The pH-dependence of the absorption changes due to the purple-to-blue transition measured at 660 nm. The data were fitted by the Henderson-Hasselbalch equation, with a pK_a of 3.6 (n = 0.9).

FIGURE 5

Kinetics of the flash-induced absorption changes in (A) AR4 and (B) BR at the wavelengths indicated (pH 6.9, 75 mM K₂SO_4, 20°C).

FIGURE 6

(A) M-formation of AR4 at the pHs indicated. (B) pH-dependence of the fraction of the fast component of M-formation of AR4. The samples were measured in gels at the given pHs. The fitted curve gives pK_a ∼9.7.

FIGURE 7

(A) pH-dependence of the normalized O-intermediate (maximum light-induced absorption change at 660 nm due to the formation of the O-intermediate divided by maximum absorption change at 410 nm due to the M-intermediate at the associated pH) in both AR4 and BR. (B) The pH-dependence of the apparent rate constants of O-formation and O-decay in AR4 (75 mM K₂SO₄, 20°C). The rate constants of O-rise and decay together with the O-amplitude were fitted with the equation assuming an unidirectional N→O→BR transition and ignoring the possibility of a N↔O back reaction (57).

FIGURE 8

Kinetics of the flash-induced absorption changes of the pH-sensitive dye pyranine at 458 nm (1) and of the O-intermediate at 660 nm (2) in a suspension of AR4 (pH 7.1, 75 mM K₂SO₄, 20°C). The two-component fit of the traces gives similar apparent time constants for the O-rise and proton uptake (1.7 ± 0.2 ms) and close time constants for O-decay (11 ms) and proton release (14 ms).

FIGURE 9

Schematic presentation of the coupling between the protonation of Asp-85 and pK_a change of the PRC in M-state. Unlike BR, the pK_a of the PRC in AR4 does not drop low enough to allow early proton release at the neutral pH of the medium due to the weakened coupling.

FIGURE 10

Amino acid sequences of (A) AR4 (2,3) and (B) AR2 (9,75). Squares and diamonds denote acidic residue and alkaline residues, respectively. The eight pairs of residues different between AR2 and AR4 are marked with large circles.