Photosynthetic Dioxygen Formation Monitored by Time-Resolved X-Ray Spectroscopy

Transcription

1 Photosynthetic Dioxygen Formation Monitored by Time-Resolved X-Ray Spectroscopy Michael Haumann and Holger Dau Freie Universität Berlin, Inst. f. Experimentalphysik, Arnimallee 14, D Berlin, Germany, Abstract. Photosynthetic water oxidation provides the dioxygen of the atmosphere. Its partial reactions proceed at a Mn 4 Ca complex bound to photosystem II of plants and cyanobacteria. Understanding the mechanism of this biological oxidation of water molecules to O 2 is one of the major challenges in life sciences. We have developed and employed X-ray absorption spectroscopy (XAS) techniques facilitating measurements on metalloenzymes at room temperature. By these techniques, we were able to resolve structural changes at the Mn ions, to follow oxidation-state changes in the microseconds time domain, and to detect a novel and likely crucial intermediate in the oxygen-evolving step of the catalytic cycle of the Mn complex. Based on the obtained results, we replace the classic S-state model of the catalytic cycle by a more elaborated reaction scheme which solves apparent inconsistencies of earlier models, explains a large body of experimental results, and provides a fresh twist in photosynthesis research. Keywords: Bioinorganic chemistry, time-resolved X-ray spectroscopy, photosynthesis, water oxidation, manganese PACS: Ht INTRODUCTION To understand, at the atomic level, the oxidation of two water molecules at the manganese-calcium complex of photosystem II (PSII) (Fig. 1) yielding the molecular oxygen that we breath, is a key challenge in biophysics research. Such knowledge likely will be of relevance for future (biomimetic) fuel production. Water oxidation is located at a pentanuclear metal complex (Mn 4 Ca) (Fig. 2) predominantly ligated by amino acid residues of the D1 protein of PSII [1]. However, due to the limited resolution of the available crystal structures (3 Å) and to X-ray induced Mn reduction during crystallography [2-4], a reliable atomic-resolution model of the Mn complex and its ligands from crystallography can not be derived yet. FIGURE 1. Schematic drawing of the photosystem II protein complex in the thylakoid membrane. Tyr-Z, tyrosine-161 of the D1 protein; P680, primary chlorophyll donor; Pheo, pheophytin; Q A,B, quinone molecules; Mn 4 Ca, catalytic metal site of water oxidation (Mn complex). The paths of electron transfer upon light excitation of PSII are indicated by solid arrows. FIGURE 2. Electron density of the Mn/Ca ions and amino acid ligands from crystallography (adapted from ref. 1). The catalytic cycle commonly is described in terms of the Kok-scheme [5] (for an extended scheme see

2 Fig. 3). It is a mayor advantage that purified PSII preparations are synchronized in the dark in the S 1 -state so that the Mn complex can be synchronously stepped through its reaction cycle by applying four consecutive nanosecond Laser flashes of visible light. The following questions need to be clarified to solve the catalytic mechanism. (1) What is the oxidation state of Mn in each of the steps of the cycle? (2) What structural changes proceed at the Mn ions? (3) What reaction intermediates are involved in the S 3 S 0 transition where O 2 ultimately is formed? MATERIALS AND METHODS PSII membrane particles with particularly high O 2 - production activity were isolated from spinach and used for the preparation of partially dehydrated samples with a concentration of ~1 mm Mn suitable for XAS [10]. X-ray absorption experiments at room temperature were carried out at the undulator beamline ID26 of the European Synchrotron Radiation Facility (ESRF) at Grenoble, France during several measuring periods. XAS spectra and transients were recorded in fluorescence mode using a photodiode or a scintillation detector and Laser-flash excitation of PSII samples in the X-ray beam. Time-resolved X-ray experiments and data evaluation were performed as previously described [7-10]. By control experiments it was verified that the extent of photoreduction of Mn during the measurements was negligible [4,10]. RESULTS AND DISCUSSION FIGURE 3. Extended version [6] of the classic Kok-scheme [5] of the S-state cycle of water oxidation. The respective transition times and the influence of ph and H/D exchange have been derived previously. The S 4 state was characterized in [7]. The S 4 state is hypothetical. The apparently irregular electron and proton transfer events can be solved in a novel reaction scheme (see Fig. 11). A well-suited tool to study these questions is X-ray absorption spectroscopy (XAS) [8,9]. It frequently has been employed to investigate enzymes in the deepfrozen state where catalysis is inactive. Of high interest are techniques which allow for investigations on metalloenzymes at room temperature (RT) where the stepping through the catalytic cycle is functional. We have developed time-resolved X-ray absorption techniques at room temperature [9] which were employed to investigate structural and oxidation state changes on each transition between the four semistable states in the catalytic cycle of the Mn complex [10]. Complete EXAFS spectra were obtained within 400 ms after S-state population by Laser flashes [10]. In X- ray experiments with 10 μs resolution we have detected a novel intermediate during the O 2 -evolving step S 3 S 0 which is formed after a deprotonation reaction prior to the reduction of Mn by electrons from water [7]. On the basis of these and further results we propose a reaction cycle of the Mn complex which involves crucial bridging-mode changes to cope with the energetic restraints of water oxidation [11]. In our study, the collection of time-resolved XAS data relies on recording of time cources of the intensity of the excited X-ray fluorescence at fixed excitation energy (timescan technique), after excitation of PSII samples situated in the X-ray beam by a series of nanosecond Laser flashes (Fig. 4). By collecting such timescans at various energies at the Mn K-edge, the construction of complete XANES and EXAFS spectra becomes feasible (sampling-xas, [9,10]). FIGURE 4. Changes of the X-ray fluorescence intensity of the Mn complex at an excitation energy of 6553 ev (around half-height of the Mn K-edge) upon the first 6 Laser flashes (arrows). A measuring interval of 20 ms around each flash is displayed; the spacing between flashes was 700 ms; about 1000 timescan traces were averaged to improve the signalto-noise ratio. The time resolution is 10 μs per data point. EXAFS Spectra of Four S-States at RT Figure 5 shows EXAFS spectra of the Mn complex after 0, 1, 2, and 3 Laser flashes predominantly

3 populating the S 1, S 2, S 3, and S 0 states, as obtained by the sampling-xas technique at RT within a time interval of only 400 ms after each flash [10]. Their spectral shape overall is similar to spectra obtained at 20 K when the S-states were populated by a flashfreeze approach [10]. This result implies that there is no temperature dependence of charge or protonation state changes at the Mn ions, for all four S-states. FIGURE 6. Bridging-mode changes between the Mn ions in the S-cycle as derived from EXAFS analysis. The fourth Mn and Ca have been omitted for clarity. For details see [10]. Oxidation State Changes: XANES at RT FIGURE 5. Fourier-transforms (FTs) of EXAFS oscillations (dots in the inset) obtained by the sampling-xas approach at RT [10] within 400 ms after S-state population by Laser flashes. FTs were calculated for 3 Å -1 k 10 Å -1. The magnitudes of the two FT peaks due to Mn-O,N (I) and Mn- Mn (II) backscattering after zero (F0) to three (F3) flashes are labeled. The lines in the inset represent simulations [10]. The following observations were extracted from Fig. 5 and interpreted in accord with previous results: (1) The magnitude of FT peak I depends on the S- state. This effect mainly reflects homogenization of the O,N distances in the first coordination sphere upon oxidation of Mn in S 0 S 1 and S 1 S 2 [10] and of the formation of (Mn IV L 6 ) 4 from (Mn IV L 6 ) 3 (Mn III L 5 ) on the S 2 S 3 transition [12]. (2) FT peak II is attributable to Mn-Mn distances of di-μ-o(h) bridged Mn pairs. Its magnitude is similar in S 1 and S 2, smaller in S 0, and largest in S 3. EXAFS simulations revealed that in S 1 and S 2 two Mn-Mn distances of ~2.7 Å, attributable to at least two Mn 2 (di-μ-o) motifs, are sufficient to account for FT peak II. In S 3, three Mn-Mn vectors with an average length of ~2.74 Å are present, suggesting the formation of a new Mn 2 (di-μ-o) motif in S 3 [10]. In S 0, the decrease of FT peak II readily is explained by the presence of two Mn-Mn distances with ~2.7 Å and ~2.85 Å length [10], the longer distance assignable to a Mn 2 (μ-oh)(μ-o) motif [10]. The changes in the bridging mode during the catalytic cycle that have been derived from EXAFS (and XANES) analysis of spectra obtained at RT and at 20 K [10] as summarized in Fig. 6. By the sampling-xas techniques, XANES spectra were obtained in four S-states (Fig. 7). FIGURE 7. XANES spectra at RT in four S-states [10]. Spectra have been obtained by the sampling-xas technique within 200 ms after the population of the respective S-state by 0 to 3 Laser flashes applied to PSII samples positioned in the X-ray beam and deconvolution of the raw spectra [10]. (1) The edge energies and shapes of the spectra are similar at RT and at 20 K [10]. Thus, the oxidation states and likely also the distribution of charges among the Mn ions are temperature-independent. (2) The position of the K-edge on the energy scale depends on the S-state; the edge energies E(i) are ordered according to E(S 0 ) < E(S 1 ) < E(S 2 ) < E(S 3 ) with an upshift of the edge position of ev occurring on each oxidizing transition as determined by the integral method [8] (which has been shown to provide an almost linear relation between edge

4 position and Mn oxidation state). Such upshifts are suggestive of Mn oxidation. (3) The edge shape differs between S 2 and S 3. The shape change, which leads to a somewhat smaller edge shift on S 2 S 3, is well explained by a change from Mn III L 5 to Mn IV L 6 on the S 2 S 3 transition [10]. Based on the analysis of experimental XANES spectra at RT and 20 K [10], on XANES simulations [12], and on comparison with model compounds [13], we propose the following oxidation states in the S-cycle: S 0, Mn III 3Mn IV 1; S 1, Mn III 2Mn IV 2; S 2, Mn III 1Mn IV 3; S 3, Mn IV 4. A particularly interesting observation was that a lag-phase is present prior to the exponential rise on the flash 3 (Fig. 8) that induces the O 2 -evolving S 3 S 0 step. At 6552 ev, its fully unambiguous interpretation is problematic. At 6556 ev, there is an isosbestic point of the XANES spectra of S 2 and S 3, so that the kinetic trace from S 3 S 0 on flash 3 is not compromised by contributions from S 2 S 3. The trace at 6556 ev again revealed a lag-phase (Fig. 9). Microsecond Resolution Studies Monitoring the transitions between the S-states by recording changes of the X-ray fluorescence at fixed excitation energies in the rise of the Mn K-edge with microsecond resolution was employed to search for intermediates [7]. Figure 8 shows Laser-flash induced X-ray transients from dark-adapted PSII samples. FIGURE 8. X-ray fluorescence transients at RT at an excitation energy of 6552 ev upon Laser flashes 1 to 4 applied to PSII samples in the X-ray beam [7]. The fitted half-rise times of the transients are indicated. About 1500 transients (from a fresh PSII sample each) have been averaged to improve the signal-to-noise ratio. Kinetic analysis of the X-ray fluorescence transients [7], which reflect shifts of the Mn K-edge on the energy scale (see Fig. 7), revealed rates of oxidation/reduction of Mn on the four S-transitions (Fig. 8) which are similar to those determined by other spectroscopic techniques. These results clearly are in favor of Mn oxidation on three S-transitions. FIGURE 9. X-ray transient on flash 3 inducing the S 3 S 0 transition at 6556 ev. The time-resolution is 10 μs per data point. (A) The lag prior to the exponential rise clearly is apparent from comparison of simulations using a single exponential ( ) or a consecutive reaction scheme [7] with two steps (line). (B) Semi-logarithmic plot, to show more clearly the lag of ~250 μs duration. Analysis of X-ray transients measured in the whole energy range of the Mn K-edge, comparison with kinetic data from chlorophyll fluorescence measurements, and taking the results from measurements on Mn model compounds [14] into account suggests [7]: During the lag phase, Mn oxidation/reduction does not occur. Instead, we propose that a deprotonation reaction happens prior to the reduction of Mn, e.g. before electrons from water molecules are transferred to Mn. The deprotonated state corresponds to the S 4 state (Fig. 10).

5 removal lead to I 8 where four electrons and four protons have been removed. (The peroxidic intermediate proposed in [15] may occur during the I 8 to I 0 transition.) The I-cycle solves the irregularities of the classic S-state model (Fig. 3). Moreover, it addresses how four successive oxidation steps can proceed without prohibitive increase in the redox potential of the Mn complex. A second mechanistic role of the four deprotonation events may be the accumulation of bases that serve as proton acceptors in the O 2 -formation step [11]. ACKNOWLEDGMENTS FIGURE 10. Tentative model for the sequence of reactions (see numbers) occurring on the O 2 -evolving transition. From the analysis of kinetic X-ray data we propose that a deprotonation (presumably of an amino acid side chain) occurs on the S 3 S 4 transition, prior to the reduction of Mn by electrons from water and to the release of dioxygen [7]. Extension of the Classic S-State Scheme Our results from XAS experiments and previous evidence suggest a novel reaction scheme of water oxidation (Fig. 11). FIGURE 11. Reaction cycle of alternating proton and electron abstraction from the Mn complex [6]. The left subscript and superscript indicate the number of accumulated oxidizing equivalents (n+, n = 0..4) and bases (m-, m = 0..4), respectively; their difference (n-m) yields the accumulated net charge of the Mn complex, which never exceeds unity. We propose that protons and electrons are removed strictly alternately from the Mn complex. Starting in I 0, eight successive steps of alternate proton and electron We thank Drs. P. Liebisch, C. Müller, and M. Grabolle and P. Loja, M. Barra, and Dr. O. Kirilenko for their contributions, Drs. T. Neisius and P. Glatzel from ESRF for excellent support, and the Deutsche Forschungsgemeinschaft (SFB498, projects C6 and C8), the BMBF, and the Volkswagen Foundation (grant I/77-575) for financial support. REFERENCES 1. B. Loll, J. Kern, W. Saenger, A. Zouni, and J. Biesiadka, Nature 438, (2005). 2. J. Yano, J. Kern, K.-D. Irrgang, M. J. Latimer, U. Bergmann, P. Glatzel, Y. Pushkar, J. Biesiadka, B. Loll, K. Sauer, J. Messinger, A. Zouni, and V. Yachandra, Proc. Natl. Acad. Sci. USA 102, (2005). 3. H. Dau, P. Liebisch, and M. Haumann, Phys. Chem. Chem. Phys. 6, (2004). 4. M. Grabolle, M. Haumann, P. Liebisch, C. Müller, and H. Dau, J. Biol. Chem. 281, (2006). 5. B. Kok, B. Forbush, and M. McGloin, Photochem. Photobiol. 11, (1970). 6. H. Dau and M. Haumann, Science 312, (2006). 7. M. Haumann, C. Müller, P. Liebisch, M. Barra, M. Grabolle, and H. Dau, Science 310, (2005). 8. H. Dau, P. Liebisch, and M. Haumann, Anal. Bioanal. Chem. 376, (2003). 9. M. Haumann, C. Müller, P. Liebisch, T. Neisius, and H. Dau, J. Synchrotron Radiat. 12, (2005). 10. M. Haumann, C. Müller, P. Liebisch, L. Iuzzolino, J. Dittmer, M. Grabolle, T. Neisius, W. Meyer-Klaucke, and H. Dau, Biochemistry 44, (2005). 11. H. Dau and M. Haumann, Photosynth. Res. 84, (2005). 12. H. Dau, P. Liebisch, and M. Haumann, Phys. Scripta T115, (2005). 13. A. Magnuson, P. Liebisch, J. Högblom, M. Anderlund, R. Lomoth, W. Meyer-Klaucke, M. Haumann, and H. Dau, J. Bioinorg. Chem. 100, (2006). 14. T. C. Weng, W. Y. Hsieh, E. S. Uffelman, S. W. Gordon-Wylie, T. J. Collins, V. L. Pecoraro, and J. E. Penner-Hahn, J. Am. Chem. Soc. 126, (2004). 15. J. Clausen and W. Junge, Nature 430, (2004).