Peter Liebisch

Photosynthetic O2 Formation Tracked by Time-Resolved X-ray Experiments

Plants and cyanobacteria produce atmospheric dioxygen from water, powered by sunlight and catalyzed by a manganese complex in photosystem II. A classic S-cycle model for oxygen evolution involves five states, but only four have been identified. The missing S4 state is particularly important because it is directly involved in dioxygen formation. Now progress comes from an x-ray technique that can monitor redox and structural changes in metal centers in real time with 10-microsecond resolution. We show that in the O2-formation step, an intermediate is formed—the enigmatic S4 state. Its creation is identified with a deprotonation process rather than the expected electron-transfer mechanism. Subsequent electron transfer would give an additional S4′ state, thus extending the fundamental S-state cycle of dioxygen formation.

Rapid Loss of Structural Motifs in the Manganese Complex of Oxygenic Photosynthesis by X-ray Irradiation at 10–300 K

Structural changes upon photoreduction caused by x-ray irradiation of the water-oxidizing tetramanganese complex of photosystem II were investigated by x-ray absorption spectroscopy at the manganese K-edge. Photoreduction was directly proportional to the x-ray dose. It was faster in the higher oxidized S2 state than in S1; seemingly the oxidizing potential of the metal site governs the rate. X-ray irradiation of the S1 state at 15 K initially caused single-electron reduction to S0* accompanied by the conversion of one di-μ-oxo bridge between manganese atoms, previously separated by ∼2.7 Å, to a mono-μ-oxo motif. Thereafter, manganese photoreduction was 100 times slower, and the biphasic increase in its rate between 10 and 300 K with a breakpoint at ∼200 K suggests that protein dynamics is rate-limiting the radical chemistry. For photoreduction at similar x-ray doses as applied in protein crystallography, halfway to the final MnII4 state the complete loss of inter-manganese distances <3Å was observed, even at 10 K, because of the destruction of μ-oxo bridges between manganese ions. These results put into question some structural attributions from recent protein crystallography data on photosystem II. It is proposed to employ controlled x-ray photoreduction in metalloprotein research for: (i) population of distinct reduced states, (ii) estimating the redox potential of buried metal centers, and (iii) research on protein dynamics.

The structure of the manganese complex of Photosystem II in its dark-stable S1-state-EXAFS results in relation to recent crystallographic data

Determination of the nuclear geometry of the Mn4Ca complex of photosystem II (PSII) may facilitate an in-depth discussion of the mechanism of photosynthetic water oxidation. The first step of structural analysis by EXAFS (extended X-ray absorption fine-structure) spectroscopy and protein crystallography is the determination of the structure of the dark-stable S1-state of the Mn complex. Approaches for normalization and simulation of EXAFS spectra are critically evaluated, in particular with respect to the accuracy of the determined coordination numbers. It is shown that the number of Mn–Mn interactions with about 2.7 A length is likely to be two in the S1-state. The presence and orientation of the 2.7 A Mn–Mn vectors as well as of the Mn–Ca distances of ∼3.3 A length is predicted by EXAFS analysis and seems to be compatible with the available crystallographic data (Zouni, Witt, J. Kern, P. Fromme, N. Kraus, W. Saenger and P. Orth, Nature, 2001, 409, Kamiya and Shen, Proc. Natl. Acad. Sci. USA, 2003, 100, 98; Ferreira, Iverson, Maghlaoui, Barber and Iwata, Science, 2004, 303, 1831). In the light of XAS results on radiation-induced modifications of the Mn4Ca complex, however, we conclude that in the course of the crystallographic data collection the Mn4Ca complex may be significantly affected by X-ray photoreduction so that the obtained electron density maps do not represent the intact complex in its S1-state. Thus, any detailed structural modelling by combination of EXAFS results and crystallographic data seems to be premature. Two motifs for the connection of Mn ions in the complex, namely either by (μ2-O)2 or (μ2-O, μ3-O) bridges, may account for Mn–Mn distances close to 2.7 A. Three basic possibilities to arrange the Mn ions of the Mn4Ca complex in its S1-state are discussed.

First steps towards time-resolved BioXAS at room temperature: state transitions of the manganese complex of oxygenic photosynthesis

Structural changes and redox transitions at the metal atoms of the active site are essential for the understanding of the catalytic mechanisms of biological metalloenzymes. First steps towards studying these processes by time-resolved X-ray absorption spectroscopy on protein samples (BioXAS) are reported. Photosystem II (PSII) catalyses the light-driven oxidation of bound water molecules at a tetranuclear manganese complex yielding the molecular oxygen of the atmosphere. In this work, first time-resolved XAS results under quasi-physiological conditions (at room temperature, non-crystalline samples) on PSII are presented. Perspectives of time-resolved BioXAS are discussed.

Linear Dichroism in the XANES of Partially Oriented Samples: Theory and Application to the Photosynthetic Manganese Complex

For molecular systems which are partially ordered with respect to one macroscopic axis, for example, the sample-surface normal, X-ray absorption spectroscopy (XAS) with linearly polarized synchrotron radiation can provide information on structure and orientation of the X-ray absorbing site (polarized or linear-dichroism XAS). Examples for such partially oriented systems are protein-carrying membrane particles deposited in the form of multilayers on surfaces, layered minerals, surface films or imperfect 2D crystals, planar electrodes or catalytic surfaces. For electric dipole transitions, equations are derived describing how partial unidirectional orientation determines the linear dichroism (LD). The approach presented facilitates description of 1) LD in multiple-scattering contributions of the extended X-ray absorption fine-structure (EXAFS) and 2) of LD in the X-ray absorption near-edge structure (LD-XANES). Structural and orientation information becomes accessible by combination with ab initio XANES calculations. The LD-XANES approach is applied to the water-oxidizing Mn complex of photosystem II. The results suggest that the (mu-O)-(mu-O) vectors of the Mn-(mu-O)(2)-Mn units are at an average angle to the membrane normal of 30-45 degrees. The best-fit structure in connection with crystallographic data suggests a specific oxidation-state assignment: Mn(1)(III)Mn(2)(III)Mn(3)(IV)Mn(4)(IV). However, currently this assignment remains uncertain. In future studies, the LD-XANES analysis should play an important role in construction of unequivocal atomic-resolution model of the photosynthetic Mn complex.