Photocycle kinetics and flash-induced proton concentration changes
Photocycle kinetics were measured at 400, 500. and 580 nm in the presence of the short-chain lipid DHPC. This lipid does not support the formation of closed bilayer vesicles, but rather forms micelles like a detergent. The time-resolved measurements showed no positive 400-nm absorbance signals at pH 8.0 or lower (Fig. 3). This is somewhat in disagreement with Béjà et al [1], who detected small 400-nm transient absorbance increases upon photolysis at pH 8.0. However, we observed a transient 400-nm absorbance increase at an elevated pH of 9.5 (fig. 3).
Figure 3  Dependence on pH of the M-like intermediate of pR. Time courses of flash-induced absorbance changes measured at 400 nm and 22°C for pR in 1% DHPC/100 mM NaCl solution at pH 6.5, 8.0 and 9.5. A positive differential absorbance at 400 nm is indicative of the presence of the M intermediate. The logarithmic time scale ranges from 100–107 μs after photolysis by a 10-ns laser pulse at 500 nm, with an energy of 3–6 mJ. At pH 9.5 in the presence of DHPC, and observing transient changes at 500 nm (fig. 4), pR undergoes a 2-phase decay after the initial unresolved absorbance decrease. Multiexponential fits show that the first decay phase has a time constant of 4 μs, in good agreement with the 4-μs rise time of the 400 nm signal (Fig. 4). The amplitude of this decay represents about 80% of the initial absorbance depletion. The second phase of the 500 nm absorbance decay occurs with a substantially slower time constant of 0.5 s, returning the remaining 20% of initial absorbance change. The slowest decay components of the positive 400-nm signal and the negative 500-nm signal follow similar kinetics, although the amplitudes of these components differ by a factor of 3. At pH 9.5, the 580 nm trace has no significant positive values indicative of an O-like intermediate, although, in agreement with earlier measurements [1], at lower pH values a red-shifted transient is the predominant positive absorbance signal (data not shown).
Figure 4  Photocycle kinetics of pR at selected wavelengths at pH 9.5. Time traces were measured at 400, 500, and 580 nm. The 400-nm trace shows the kinetics of the M intermediate, i.e. the deprotonated Schiff base, as in Fig. 3. The 500-nm trace shows the depletion signal of pR at the earliest times, and then the time course of the N intermediate as well as return of the pR resting state. The 580-nm trace is indicative of an O-like intermediate. The conditions are 1% DHPC, 100 mM NaCl, pH9.5 at 22°C. The laser excitation is as in fig. 3. Figure 5 shows a different type of time-resolved measurement, probing not the pR chromophore, but rather pH changes in the protein environment. Proton concentration changes in the aqueous bulk phase were measured with the pH sensitive dye cresol red, which has a pKa of 8.2–8.5. The bottom trace in Figure 5 shows the absorbance change of the indicator during the pR photocycle. The negative signal is indicative of a pH decrease, corresponding to transient H+ release from the protein into the solution. The best-fit time constant for the release phase is 6 μs. The positive 400 nm trace in fig. 5 (reproduced from fig. 3) shows that the proton release and uptake follow kinetics very similar to the apparent formation and decay of M, as is typically seen in bR near neutral pH [6,7,21]. However, no proton release signal could be observed for pR at pH 6 or 8 (data not shown).
Figure 5  Comparison of the kinetics of M formation and decay with kinetics of ET release and uptake. The time trace of the M-like intermediate was measured at 400 nm (upper panel). Time-resolved H+ concentration changes (lower panel) were measured with the pH indicator dye Cresol Red. A negative Cresol Red absorbance change at 580 nm is indicative of a transient decrease in the pH of the solution, i.e. of H+ release by pR. Solid lines represent multiexponential fits, with the main rise and decay times indicated for the M intermediate. The H+ release and uptake time constants obtained from the fit are marked with arrows pointing down for release and pointing up for uptake. Sample and excitation conditions are as in fig. 4.