Kenneth Sauer

A Spectroscopic Study of a Photosystem I Antenna Complex

The unusual long wavelength fluorescent behavior of Photosystem I (PSI) has been well documented (1) This study investigates the origin and temperature dependence of this emission using an intact peripheral antenna preparation isolated from a PSI complex originally extracted from spinach. This antenna complex (LHCP-I) contains polypeptides in the 19–24 kDa range and exhibits a red shift in emission maximum from 685nm (F685) to 735nm (F735) as the temperature is lowered. Fluorescence excitation spectra demonstrate that chlorophyll b (chl b) preferentially stimulates the long wavelength emission at both room temperature and 77K and also indicate the presence of a long wavelength absorber in the 703–708nm range. Excitation polarization scans show a rising polarization value reaching a maximum of 0.3 from 705nm to 725nm, confirming the presence of a long wavelength pigment or pigments which are primarily responsible for emission at 735nm (F735). Absorption spectra demonstrate that this species comprises a small percentage of the total pigment population in the light harvesting antenna. The overall fluorescence yield of the complex increases as the temperature is lowered suggesting the presence of a quenching mechanism which does not involve the reaction center, P700.

Calcium and Chloride Cofactors of the Oxygen Evolving Complex - X-Ray Absorption Spectroscopy Evidence for A Mn/Ca/Cl Heteronuclear Cluster

The oxygen-evolving complex (OEC) of photosystem II (PSII) in green plants and algae contains a cluster of four Mn atoms in the active site, which catalyzes the oxidation of water to dioxygen. Along with Mn, Cl− and Ca2+ are essential cofactors for oxygen evolution (1).

Light-Induced Changes in X-Ray Absorption (K-Edge) Energies of Manganese in Photosynthetic Membranes

This report describes our observations of the participation of manganese in electron transport and oxidation equivalent storage by the photosynthetic oxygen evolution complex of higher plants. The oxidizing potential produced by photosynthetic charge separation is stored near the site of water oxidation in a membrane-bound complex that contains manganese. Broken, washed chloroplast preparations typically contain 4–6 Mn per PSII reaction center, and approximately 2/3 of this quantity is released by mild treatments that specifically inactivate O2 evolution [1– 3]. Recent studies with inside out thylakoids [4] and photosystem II (PSII) preparations [5] have given rise to a reevaluation of the Mn content per photosynthetic unit, but it remains clear that a direct relation exists between Mn and O2 evolution.

Chloride ligation in inorganic manganese model compounds relevant to Photosystem II studied using X-ray absorption spectroscopy

Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OEC) of Photosystem II (PS II). Although proposed to be directly ligated to the Mn cluster of the OEC, the specific structural and mechanistic roles of chloride remain unresolved. This study utilizes X-ray absorption spectroscopy (XAS) to characterize the Mn–Cl interaction in inorganic compounds that contain structural motifs similar to those proposed for the OEC. Three sets of model compounds are examined; they possess core structures MnIV3O4X (X=Cl, F, or OH) that contain a di-μ-oxo and two mono-μ-oxo bridges or MnIV2O2X (X=Cl, F, OH, OAc) that contain a di-μ-oxo bridge. Each set of compounds is examined for changes in the XAS spectra that are attributable to the replacement of a terminal OH or F ligand, or bridging OAc ligand, by a terminal Cl ligand. The X-ray absorption near edge structure (XANES) shows changes in the spectra on replacement of OH, OAc, or F by Cl ligands that are indicative of the overall charge of the metal atom and are consistent with the electronegativity of the ligand atom. Fourier transforms (FTs) of the extended X-ray absorption fine structure (EXAFS) spectra reveal a feature that is present only in compounds where chloride is directly ligated to Mn. These FT features were simulated using various calculated Mn–X interactions (X=O, N, Cl, F), and the best fits were found when a Mn–Cl interaction at a 2.2–2.3xa0Å bond distance was included. There are very few high-valent Mn halide complexes that have been synthesized, and it is important to make such a comparative study of the XANES and EXAFS spectra because they have the potential for providing information about the possible presence or absence of halide ligation to the Mn cluster in PS II.

Refined Model of the Oxidation States and Structures of the Mn/Ca/Cl Cluster of the Oxygen Evolving Complex of Photosystem II

Central to the problem of photosynthetic oxygen evolution is the structure and function of the Mn/Ca/Cl complex that appears to be the locus of charge accumulation and water splitting. In the recent past our group has presented a topological model for the structure of the tetranuclear Mn cluster, the oxidation state assignments of the S-states of the Kok cycle, the orientation of the Mn-Mn vectors relative to the membrane normal, and evidence for the proximity of Ca to the Mn (1–3).

Recent Advances Toward A Structural Model for the Photosynthetic Oxygen-Evolving Manganese Cluster

Photosynthetic water oxidation occurs in the oxygen evolving complex (OEC) of photosystem II (PS II). One-electron photo-oxidations in the reaction center of PS II are coupled to the four-electron oxidation of water in the OEC. As the PS II reaction center sequentially extracts electrons, the OEC cycles through five intermediate oxidation states (So-S4) where So is the least oxidized and S4 is a transient state which decays to So with the release of a dioxygen molecule. A complex of four manganese atoms has been shown to function in charge accumulation and is thought to form the catalytic site for the water oxidation reaction. The structure of this manganese complex and the mechanism of water oxidation have been the subject of vigorous inquiry by a great many research groups (Sauer et al., 1992).

Protein Sequence Homologies between Portions of the L and M Subunits of Reaction Centers of Rhodopseudomonas capsulata and the 32 KD Herbicide-binding Polypeptide of Chloroplast Thylakoid Membranes and a Proposed Relation to Quinone-binding Sites

The sequences of the reaction center polypeptides of Rhodopseudomonas capsulata have been determined (D.C. Youvan, M. Alberti, H. Begusch, E.J. Bylina and J.E. Hearst, these proceedings, 1983). We have discovered that there is a highly conserved pattern of sequences of amino acids which is common to the L and M subunits of R. capsulata and the 32 kd herbicide-binding polypeptide of chloroplast thylakoid membranes in spinach and tobacco. This conservation of sequence has survived since the divergence of these organisms, which is estimated to have taken place 3 billion years ago (J.M. Olson, 1981). Such a striking conservation of amino acid sequence suggests that these portions of all three proteins, which are approximately 60% in from the amino terminus in each case, are at the functional centers of these proteins. Because the R. capsulata reaction centers are known to contain two bound quinones (J.R. Bowyer et al., 1979), because herbicide binding to the 32 kd thylakoid membrane protein is known to block quinone binding to photosystem 2 (B.R. Velthuys, 1981; C.A. Wraight, 1981), and because quinones are known to have unique importance to the primary chemical events in bacterial reaction centers as well as in photosystem 2 of chloroplasts, it is our hypothesis that this highly conserved sequence of amino acids is involved in quinone binding and function.

Photosystem II and Water Oxidation

The relation of the structure and organization of the Photosystem II reaction centers to those from Photosystem I or from the green or purple bacteria presents an interesting example of comparative biochemistry. Similarities between PS II and purple bacterial reaction centers include aspects of the reaction center proteins, the stoichiometry of chlorophyll and pheophytin in the reaction center and the complex of iron with quinones as the primary electron acceptor. In each of these respects the reaction centers of PS I or green bacteria, however, have no obvious similarity.

Structures and Oxidation States of MN in Several S-States of Photosystem II Determined by X-Ray Absorption Spectroscopy

X-ray spectroscopy employing synchrotron radiation has been applied to the study of the Mn clusters associated with the oxygen evolving complex (OEC) of Photosystem II. We have shown earlier that the edge energies can be used to indicate oxidation state(s) and the EXAFS used to determine the local structure about Mn. [1a, b] Since our first use this method both the quality of the biological preparations and the physical methodology have improved sufficiently to permit studies of PS II membranes in four of the five S-states postulated by Kok. (2]. Here we present a brief resume of our results on samples in the dark adapted S1 state, the S2 state formed by illumination at 190K, the S3 state formed by a double turnover [3a,c] and an S0-like state formed by illumination of dark adapted samples containing 40–60 uM hyudroxylamine. [3b,c]

Parallel Polarization EPR Studies of the Oxygen-Evolving Complex of Photosystem II

The oxygen-evolving complex of Photosystem II mediates the oxidation of water to molecular oxygen. According to a model devised by Kok and co-workers (1), the complex couples the four-electron water oxidation process to the single-electron primary photochemistry by cycling through a series of states, S0 – S4, as it transfers electrons to reduce the photo-oxidized primary donor. Four manganese ions are thought to be required for oxygen evolution activity, but the structural organization of the manganese ions and their oxidation states throughout the S-state cycle remain in question. Although the system has been thoroughly studied with conventional EPR, only two signals attributed to manganese have been reported, and both occur in the S2 state. The multiline signal, comprised of a number of hyperfine components centered near g=2, is consistent with an exchange-coupled mixed-valence manganese cluster (2). A second signal appears at an effective g value of 4.1 (3,4), and the correlation of the generation of this signal with an increase in the manganese X-ray absorption energy implies that the signal may originate from manganese (5). Recently proposed models for the structural organization of manganese and the origin of the S2 state EPR signals include the suggestion that the signals arise from different spin states of a single tetranuclear cluster (6), or that the signals arise from separate centers: for example, that the multiline signal is due to a mixed-valence binuclear cluster and the g=4.1 signal originates from a mononuclear Mn(IV) site (7).