Enthalpy changes during photosynthetic water oxidation tracked by time-resolved calorimetry using a photothermal beam deflection technique

Abstract

The energetics of the individual reaction steps in the catalytic cycle of photosynthetic water oxidation at the Mn(4)Ca complex of photosystem II (PSII) are of prime interest. We studied the electron transfer reactions in oxygen-evolving PSII membrane particles from spinach by a photothermal beam deflection technique, allowing for time-resolved calorimetry in the micro- to millisecond domain. For an ideal quantum yield of 100%, the enthalpy change, DeltaH, coupled to the formation of the radical pair Y(Z)(.+)Q(A)(-) (where Y(Z) is Tyr-161 of the D1 subunit of PSII) is estimated as -820 +/- 250 meV. For a lower quantum yield of 70%, the enthalpy change is estimated to be -400 +/- 250 meV. The observed nonthermal signal possibly is due to a contraction of the PSII protein volume (apparent DeltaV of about -13 A(3)). For the first time, the enthalpy change of the O(2)-evolving transition of the S-state cycle was monitored directly. Surprisingly, the reaction is only slightly exergonic. A value of DeltaH(S(3)-->S(0)) of -210 meV is estimated, but also an enthalpy change of zero is within the error range. A prominent nonthermal photothermal beam deflection signal (apparent DeltaV of about +42 A(3)) may reflect O(2) and proton release from the manganese complex, but also reorganization of the protein matrix.

Figure 1

Extended reaction scheme of the S-state cycle of photosynthetic water oxidation at the Mn complex of PSII (9–12). The cycle comprises eight steps of alternate electron and proton removal from the Mn complex. The ${\text{S}}_3^{\text{n}}$ state corresponds to the S₄ state described in ; ${\text{S}}_4^{+}$ is a hypothetical intermediate where four protons and four electrons have been abstracted from the Mn complex before O₂ release. Within <1 μs after each flash, ${\text{Y}}_{\text{Z}}^{\cdot +}$ is formed. The halftimes of the transitions between the semistable S states (bold characters) were taken from . The superscripts “n” (neutral) and “+” (one positive charge) denote the overall charge of the Mn complex resulting from the sum of electron abstraction (+1/e⁻) and proton release (−1/H⁺) up to the respective S state. For a detailed discussion of the reaction cycle, see (9–12).

Figure 2

Photothermal beam deflection (PBD) experiment. (A) Schematic drawing of the experimental setup in our laboratory. Lenses for beam shaping are omitted for clarity. Excitation of the sample by a pump-laser flash (532 nm) and subsequent heat release and nonthermal changes in the sample lead to the deflection of the continuous-wave probe beam (920 nm) by an angle ϕ, resulting in the PBD signal. (B) Normalized intensity profiles and relative positioning of pump- and probe-laser beams as measured by a photodiode in the center of the sample cuvette. The FWHM values are 0.26 mm (920 nm) and 0.54 mm (532 nm). (C) PBD signal of the calorimetric standard BCP; 1000 transients have been averaged. (Upper inset) The signal rise time of ∼3 μs is limited by the differential-photodiode detector. (Lower inset) Within 20 ms after the exciting flash, the signal decays by only ∼3% of its initial amplitude.

Figure 3

Absorption spectra of a PSII membrane sample, the dye BCP, a mixture of PSII and BCP, and the buffer. That the difference spectrum (PSII + BCP) - BCP is identical to that of PSII alone indicates that the spectrum of PSII was unchanged in the presence of BCP, and vice versa. The pump- and probe-laser wavelengths are indicated by arrows. From the spectra of BCP and PSII, the background absorption of the buffer was subtracted. Spectra of solutions containing PSII additionally were corrected for scattering contributions. From the spectra of PSII and BCP, the absorption (A_exc) at the excitation wavelength (532 nm) was determined.

Figure 4

PBD signals of PSII membranes for repetitive pump-flash excitation. About 1000 signals from five samples were averaged; the time resolution was 5 μs/data point (E_exc = 6 μJ, T = 20°C). A standard PSII sample (20 μg Chl/mL, 20 μM DCBQ) was used as the control (upper trace). The slow phase (X_ms) was simulated (line) using an exponential function with t_1/2 = 1.15 ms. (Middle trace) Sample with 40 μM DCMU + 20 μM DCBQ. The small and rapid signal decay after the exciting flash may reflect a contribution due to the ${\text{S}}_1^{\text{n}}\to S_2^{+}$ transition. (Lower trace) Sample with 40 μM DCMU and 20 μM DCBQ, with additional continuous illumination.

Figure 5

(A) PBD signals induced by the exciting pump flash (arrows) after the application, preceding the pump flash, of zero to seven saturating flashes to initially dark-adapted PSII membranes. E_exc = 8.5 μJ, ∼200 transients were averaged, the time resolution was 50 μs/data point, and T = 20°C. The rising signals in the millisecond time range (X_ms) were simulated using a t_1/2 of 1.15 ms, as exemplified for the transient on the third flash (line). (B) The amplitude of X_ms (circles) as a function of the total flash number (saturating flashes plus pump flash). The solid line represents a simulation using the Kok model and 10% of misses and 100% of centers in state ${\text{S}}_1^{\text{n}}$ before light-flash application.

Figure 6

PBD signals of PSII membranes at three pump-flash energies (at 20°C). The slow phase (X_ms) of the three signals was simulated using an exponential function with t_1/2 = 1.15 ms. Note the increase of X_prompt (dashes) relative to X_ms at increasing E_exc.

Figure 7

Dependence of the PBD signals of PSII on the energy (E_exc) of the pump flash. (A) Relative magnitudes of X_ms (solid circles) reveal single-exponential saturation behavior (line). (B) Relative magnitudes of PBD signals (X_prompt/E_exc) due to instantaneous heat release. X_prompts are from data in Figs. 4 (+DCMU) (open circles) and 6 (solid circles). Data points were simulated (line) using Eq. 9 ($E_{\text{exc}}^{1/2}=4.0\pm 0.6\mu \text{J},x^{max}=5.95\mu \text{J}$). Solid triangles represent X_prompt from data in Fig. 4 (+DCMU + light). The dashed line accounts for the conversion of 100% of the absorbed excitation energy into heat.

Figure 8

(A) PBD signals of PSII membranes for repetitive flash excitation in the absence (upper trace) and presence (middle trace) of BCP (6.8 μg/mL, similar absorption of 0.14 at 532 nm to that of PSII membranes); the lower trace represents the difference signal. About 1000 transients were averaged; the time resolution was 10 μs/data point (E_exc = 5 μJ, T = 20°C). A signal of BCP in buffer (triangles) scaled to the magnitude of that resulting from the measurement in the presence of PSII is shown for comparison (lower trace). (B) X_prompt from measurements similar to those in A of BCP and PSII membranes at increasing absorption at 532 nm determined from spectra shown in Fig. 3. The dashed line denotes the absorption (A_exc) of 0.14 of the PSII membranes at a Chl concentration of 20 μg/mL.

Figure 9

PBD signals of PSII at the indicated excitation energies and buffer temperatures. About 1000 transients at 10 μs/data point (5 μJ) and 5000 transients at 20 μs/data point (1.2 μJ) were averaged. X_ms (dashed lines) was simulated (solid line) using t_1/2 values of 0.77 ms (32.5°C), 1.45 ms (12.5°C), and 2.20 ms (−0.1°C) at E_exc = 5 μJ, and of 0.79 ms (30°C) and 2.27 ms (−1°C), respectively, at E_exc = 1.2 μJ. Note that the magnitude of X_ms is similar in all traces, whereas X_prompt strongly decreases with decreasing temperature.

Figure 10

Arrhenius plot of the rate constant of the millisecond phase (X_ms) of PBD signals due to the oxygen-evolving transition. Data points were derived from measurements at E_exc = 5 μJ similar to those in Fig. 9left, and slightly smoothed by adjacent averaging over two points for display. The line represents a fit using the Arrhenius equation and an activation energy (E_a) of 230 meV (k = k₀(exp(−E_a/k_BT)); k_B is the Boltzmann constant, and the value of the preexponential factor, k₀, was 5.75 × 10³ ms⁻¹).

Figure 11

Temperature dependence of X_prompt (left) and X_ms (right) of PSII (solid circles) compared to the PBD signal of the standard BCP (triangles) at three excitation energies (for equal absorption at 532 nm). Bold lines represent fit curves calculated using Eqs. 4 (BCP) and 5 (PSII) and the parameters shown in Table 1. PBD signals of BCP were measured in the presence (open triangles) or absence (solid triangles) of PSII membranes. At T₀ = −16 ± 1°C (dotted lines), the thermal contributions to the PBD signal vanish and only the ΔN-related signals remain. ΔN is negative for ${\text{Y}}_{\text{Z}}^{\cdot +}{\text{Q}}_{\text{A}}^{-}$ formation (X_prompt), but positive for the O₂-evolving transition (X_ms). From the fit curves, the magnitudes of ΔN, Q_St, and ΔQ (arrows) in Table 1 were determined. In the middle chart in the left column, the amplitudes of PBD signals of BCP measured in pure water (+) and a respective simulation yielding $T_0^0{=0}^{\circ }\text{C}$ (thin line) are shown for comparison.

Figure 12

Fractions of the absorbed energy of a 532-nm photon that are released as heat (ΔQ) from PSII upon ${\text{Y}}_{\text{Z}}^{\cdot +}{\text{Q}}_{\text{A}}^{-}$ formation (solid circles, left y axis) and upon the O₂-evolving transition (open squares, right y axis) plotted versus the excitation energy. Data points correspond to the values listed in Table 1. Curves represent fits to the data (solid line, ΔQ_prompt; dashed line, ΔQ_ms) using a saturation behavior as described by Eq. 9 and a value of $E_{\text{exc}}^{1/2}$ of 4 μJ (derived from X_prompt (see Fig. 7 B)). Dotted lines denote the ΔQ values from extrapolations of the simulation curves to E_exc = 0.