Photosynthetic water oxidation at elevated dioxygen partial pressure monitored by time-resolved X-ray absorption measurements - PubMed (original) (raw)

Photosynthetic water oxidation at elevated dioxygen partial pressure monitored by time-resolved X-ray absorption measurements

Michael Haumann et al. Proc Natl Acad Sci U S A. 2008.

Abstract

The atmospheric dioxygen (O(2)) is produced at a tetramanganese complex bound to the proteins of photosystem II (PSII). To investigate product inhibition at elevated oxygen partial pressure (pO(2) ranging from 0.2 to 16 bar), we monitored specifically the redox reactions of the Mn complex in its catalytic S-state cycle by rapid-scan and time-resolved X-ray absorption near-edge spectroscopy (XANES) at the Mn K-edge. By using a pressure cell for X-ray measurements after laser-flash excitation of PSII particles, we found a clear pO(2) influence on the redox reactions of the Mn complex, with a similar half-effect pressure as determined (2-3 bar). However, XANES spectra and the time courses of the X-ray fluorescence collected with microsecond resolution suggested that the O(2) evolution transition itself (S(3)-->S(0)+O(2)) was not affected. Additional (nonstandard) oxidation of the Mn complex at high pO(2) explains our experimental findings more readily. Our results suggest that photosynthesis at ambient conditions is not limited by product inhibition of the O(2) formation step.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Hypothetical energy-level diagram for a thermodynamic O2 backpressure effect on the S4→S0 transition. The Gibbs free energy, _G_P, of the final product state (S0 + O2) increases by ≈60 meV per 10-fold _p_O2 increase. At _p_O2 > 10 bar, the energy of a peroxidic reaction intermediate was predicted to be higher than _G_P, implying almost complete inhibition of O2 formation (10). The energy of the S2* reaction intermediate has been proposed to be above (10) or below (11) the initial YZoxS3-state. In the present work, validity of the above scheme is scrutinized and eventually rejected.

Fig. 2.

Fig. 2.

XANES spectra of the Mn complex measured after the application of 0–4 laser flashes to PSII samples at _p_O2 of 0.2 bar (A) and 11 bar (B). Spectra are the average of 2 (0.2 bar) and 4 (11 bar) sets of independently measured spectra. (Insets) Expanded half-height region of the K-edges. (C) Calculated spectra of pure S-states (color code as indicated), obtained by deconvolution of the spectra in A and B using parameters listed in Table 1 [lines, 0.2 bar; triangles, 11 bar O2 (a); dots, 11 bar O2 (b)] and in the legend of Fig. 3. (Inset) Corresponding difference spectra.

Fig. 3.

Fig. 3.

Mn K-edge energies (filled circles) determined from spectra as in Fig. 2 (A, 0.2 bar; B, 11 bar O2). Error bars represent SD values from 2 (at 0.2 bar) and 4 (at 11 bar) datasets. The lines were obtained by simulation of the flash patterns using the parameters listed in Table 1 [in B: solid line (a), dashed line (b)]. An S1-state edge energy of 6,551.5 eV and edge shifts as determined in (27, 28) were used in the simulations. (C and D) Filled circles represent the magnitude of the millisecond rise in the X-ray absorption at 6,552.5 eV (Fig. 4) caused by Mn reduction in the S3→S4→S0 transition. Edge energy estimates derived from the X-ray transients in Fig. 4 are shown for comparison in A and B (open triangles, assuming direct proportionality between edge shift and X-ray fluorescence change for excitation at 6,552.5 eV; normalization to equal first-flash response). Lines in C and D show simulations with parameters in Table 1 [in D: (a) solid line, (b) dashed line].

Fig. 4.

Fig. 4.

Time courses of the X-ray fluorescence at _p_O2 of 0.2 bar (Left) and 13 bar (Right) as induced by the first 8 of a train of 10 laser flashes applied to dark-adapted PSII samples (excitation energy of 6,552.5 eV). Approximately 500 (0.2 bar) and 1,500 (13 bar) transients were averaged and are displayed at a resolution of 50 μs per data point. The 13 bar transients were obtained by averaging measurements for _p_O2 values ranging from 11 to 16 bar. Data were normalized such that the amplitude of the response to the 1st flash was similar for the 0.2 and the 13 bar datasets. Smooth lines represent simulations by a biexponential function using a halftime of 1.2 ms to account for Mn reduction (positively directed changes) and variable halftimes for Mn oxidation (negatively directed signals). For selected transients the contributions from Mn oxidation (open arrows) and Mn reduction (filled arrows) are indicated.

Fig. 5.

Fig. 5.

Half-effect O2 pressure and rate of Mn reduction. (A) Pressure dependence of the O2 effect on PSII and (B) averaged X-ray transients collected at 0.2 and 13 bar. (A) Amplitudes of the millisecond phases were determined for the X-ray transients induced by the 2nd and 3rd flashes; the ratio of these amplitudes is shown (filled circles; error bars denote SD). The line represents an asymptotic simulation with a _p_O21/2 of 2.9 bar. (B) Transients represent the average of the transients on the first 10 flashes applied to dark-adapted PSII. Smooth lines denote biexponential simulations. The signal rise associated with Mn reduction in the S3→S4→S0 transition was simulated with an identical halftime of 1.2 ms for transients collected at _p_O2 of 0.2 and 13 bar. The initial decrease was approximated by an exponential function with halftimes of Mn oxidation of 105 ± 20 μs (0.2 bar) and 120 ± 20 μs (13 bar).

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