Towards a spin coupling model for the Mn4 cluster in Photosystem II

The X-band EPR spectra of the IR sensitive untreated PSII and of MeOH- and NH₃-treated PSII from spinach in the S₂-state are simulated with collinear and rhombic g- and Mn-hyperfine tensors. The obtained principal values indicate a 1Mn(III)3Mn(IV) composition for the Mn₄ cluster. The four isotropic components of the Mn-hyperfine tensors are found in good agreement with the previously published values determined from EPR and ⁵⁵Mn-ENDOR data. Assuming intrinsic isotropic components of the Mn-hyperfine interactions identical to those of the Mn-catalase, spin density values are calculated. A Y-shape 4J-coupling scheme is explored to reproduce the spin densities for the untreated PSII. All the required criteria such as a S=1/2 ground state with a low lying excited spin state (30 cm⁻¹) and an easy conversion to a S=5/2 system responsible for the g=4.1 EPR signal are shown to be satisfied with four antiferromagnetic interactions lying between −290 and −130 cm⁻¹.     

Theoretical study of the multiline EPR signal from the S2 state of the oxygen evolving complex of photosystem II: Evidence for a magnetic tetramer

The Oxygen evolving complex of plant photosystem II is made of a manganese cluster that gives rise to a low temperature EPR multiline signal in the S₂ oxidation state. The origin of this EPR signal has been addressed with respect to the question of the magnetic couplings between the electron and nuclear spins of the four possible Mn ions that make up this complex. Considering Mn(III) and Mn(IV) as the only possible oxidation states present in the S₂ state, and no large anisotropy of the magnetic tensors, the breadths of the EPR spectra calculated for dimers and trimers with S = ½ have been compared with that of the biological site. It is concluded that neither a dinuclear nor a trinuclear complex made of Mn(III) and Mn(IV) can be responsible for the multiline signal; but that, by contrast, a tetranuclear Mn complex can be the origin of this signal. The general shape of the experimental spectrum, its particular hyperfine pattern, the positions of most of the hyperfine lines and their relative intensities can be fit by a tetramer model described by the following six fitting parameters: g ≈ 1.987, A₁ ≈ 122.4 10^-4 cm^-1, A₂ ≈ 87.2 10^-4 cm^-1, A₃ ≈ 81.6 10^-4 cm^-1, A₄ ≈ 19.1 10^-4 cm^-1 and δH = 24.5 G. A second model described by parameters very close to those given above except for A₄ ≈ 77.5 10^-4 cm^-1 gives an equally good fit. However, no other set of parameters gives an EPR spectrum that reproduces the hyperfine pattern of the S₂ multiline signal. This demonstrates that in the S₂ state of the oxygen evolving complex, the four manganese ions are organized in a magnetic tetramer.     

Water binding to the oxygen-evolving system of chloroplasts; effects of isotope substitution on the S2 state EPR spectrum

Replacement of H₂O by ²H₂O in oxygen-evolving Photosystem II preparations caused an increased resolution of the fine structure of the S₂ state EPR spectrum. In both ²H₂O and H₂O samples, comparison of the S₂ spectra generated by illumination at 200 and 283 K (10°C) showed a difference in the fine structure on the hyperfine lines. A reduction in the spacing of the outer hyperfine lines was also observed when samples illuminated at 283 K were compared to those where S₂ was formed by 200 K illumination. The observations are interpreted as due to proton binding, perhaps as water, at or near the manganese complex giving rise to the S₂ signal.     

Absorbance difference spectra of the successive redox states of the oxygen-evolving apparatus of photosynthesis

The spectra of the absorbance changes due to the turnover of the so-called S-states of the oxygen-evolving apparatus were determined. The changes were induced by a series of saturating flashes in dark-adapted Photosystem II preparations, isolated from spinach chloroplasts. The electron acceptor was 2,5-dichloro-p-benzoquinone. The fraction of System II centers involved in each S-state transition on each flash was calculated from the oscillation pattern of the 1 ms absorbance transient which accompanies oxygen release. The difference spectrum associated with each S-state transition was then calculated from the observed flash-induced difference spectra. The spectra were found to contain a contribution by electron transfer at the acceptor side, which oscillated during the flash series approximately with a periodicity of two and was apparently modulated to some extent by the redox state of the donor side. At the donor side, the S₀ → S₁, S₁ → S₂ and S₂ → S₃ transitions were all three accompanied by the same absorbance difference spectrum, attributed previously to an oxidation of Mn(III) to Mn(IV) (Dekker, J.P., Van Gorkom, H.J., Brok, M. and Ouwehand, L. (1984) Biochim. Biophys. Acta 764, 301–309). It is concluded that each of these S-state transitions involves the oxidation of an Mn(III) to Mn(IV). The spectrum and amplitude of the millisecond transient were in agreement with its assignment to the reduction of the oxidized secondary donor Z⁺ and the three Mn(IV) ions.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S₁-to-S₂ transition was obtained with Ca²⁺-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca²⁺ to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S₂/S₁ FTIR spectrum upon Ca²⁺ depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca²⁺ depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K⁺ at the Ca²⁺ site. In the present study, the preparation of the Ca²⁺-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K⁺. The obtained S₂/S₁ spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm^− 1 and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K⁺. This result indicates that the drastic FTIR changes are a pure effect of Ca²⁺ depletion, and provides solid evidence for the general view that Ca²⁺ is strongly coupled with the Mn cluster.     

Review of NMR and μSR studies in the molecular nanomagnet Mn12-ac

We present a comprehensive review of the NMR and μSR studies performed in the molecular nanomagnet Mn12 a system characterized and widely studied by Prof. Gatteschi’s group in Florence. The proton (¹H, ²D) NMR, the ⁵⁵Mn NMR and the μSR investigations have yielded important information regarding both static and dynamic magnetic properties of the molecule. The magnetic and quadrupole hyperfine interactions have been extracted from NMR data. The spin dynamics at high and intermediate temperature associated with the zero dimensionality and with the spin–phonon coupling has been studied together with the spin dynamics in the quantum tunneling low temperature regime. The local spin configuration in the giant S = 10 total spin ground state has been determined via ⁵⁵Mn NMR in zero magnetic field and with fields parallel and perpendicular to the anisotropy axis. Finally a novel method is described to monitor the relaxation of the magnetization from the time evolution of the NMR spectrum.     

Specific loss of the extrinsic 18 KDa protein from Photosystem II upon heating to 47 °C causes inactivation of oxygen evolution likely due to Ca release from the Mn-complex

Exposure of Photosystem II (PS II) membrane particles from spinach to a temperature of 47 °C caused the rapid release of the 18 kDa protein in parallel to inactivation of oxygen evolution. Previously, it has been suggested that the first heat-jump response involves rapid Ca release from the Mn complex of O₂-evolution, followed by the slower release of (2 + 2) Mn^II ions [Pospisil P et al. (2003) Biophys J 84: 1370–1386]. Here, the predicted biphasic Mn^II release to the bulk was verified by atomic absorption spectroscopy (AAS). Analysis of laser flash-induced delayed fluorescence transients suggests that the loss of the essential Ca ion from the Mn₄Ca complex in the dark is due to the loss of the 18 kDa protein. The S₂-state multiline EPR signal of the Mn complex was still generated in heat-treated PS II presumably lacking Ca, but retaining four Mn ions.Dedicated to Professor Norio Murata on the occasion of his retirement     

EPR and ENDOR studies of the water oxidizing complex of Photosystem II

A comparative study of X-band EPR and ENDOR of the S₂ state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S₂ state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S₂ state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. ¹H-ENDOR experiments performed at two different field positions of the S₂ state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382–2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D₂O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S₂.Abbreviations cw continuous wave - ENDOR electron nuclear double resonance - EPR electron paramagnetic resonance - hf hyperfine - hfc hyperfine coupling - MLS multiline signal - PS II Photosystem II - rf radio frequency - WOC water oxidizing complex     

Oxidation state changes of the Mn<Subscript>4</Subscript>Ca cluster in Photosystem II

A detailed electronic structure of the Mn₄Ca cluster is required before two key questions for understanding the mechanism of photosynthetic water oxidation can be addressed. They are whether all four oxidizing equivalents necessary to oxidize water to O₂ accumulate on the four Mn ions of the oxygen-evolving complex, or do some ligand-centered oxidations take place before the formation and release of O₂ during the S₃ → [S₄] → S₀ transition, and what are the oxidation state assignments for the Mn during S-state advancement. X-ray absorption and emission spectroscopy of Mn, including the newly introduced resonant inelastic X-ray scattering spectroscopy have been used to address these questions. The present state of understanding of the electronic structure and oxidation state changes of the Mn₄Ca cluster in all the S-states, particularly in the S₂ to S₃ transition, derived from these techniques is described in this review.     

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S₀ and S₂ states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D₂O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S₀ and S₂ state multiline disappeared after 3 h of D₂O incubation in the S₀ and S₁ states, respectively. The signal with 4.0 MHz hyperfine constants in S₀ state multiline disappeared after 3 h of D₂O incubation in the S₀ state, while the similar signal in S₂ state multiline disappeared only after 24 h of D₂O incubation in the S₁ state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 Å, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.     

The tetra-manganese complex of photosystem II during its redox cycle – X-ray absorption results and mechanistic implications

Using X-ray absorption spectroscopy (XAS), relevant information on structure and oxidation state of the water-oxidizing Mn complex of photosystem II has been obtained for all four semi-stable intermediate states of its catalytic cycle. We summarize our recent XAS results and discuss their mechanistic implications. The following aspects are covered: (a) information content of X-ray spectra (pre-edge feature, edge position, extended X-ray absorption fine-structure (EXAFS), dichroism in the EXAFS of partially oriented samples); (b) S₁-state structure; (c) X-ray edge results on oxidation state changes; (d) EXAFS results on structural changes during the S-state cycle; (e) a structural model for the Mn complex in its S₃-state; (f) XAS-based working model for the S₂–S₃ transition; (g) XAS-based working model for the S₀–S₁ transition; (h) potential role of hydrogen atom abstraction by the Mn complex. Finally, we present a specific hypothesis on the mechanism of dioxygen formation during the S₃–(S₄)–S₀ transition. According to this hypothesis, water oxidation is facilitated by manganese reduction that is coupled to proton transfer from a substrate water to bridging oxides.     

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution: II. A binuclear manganese-containing 34 kilodalton protein,a probable component of the water dehydrogenase enzyme

Extraction conditions have been found which result in the retention of managanese to the 33–34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228–236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33–34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O₂-evolving, detergent-extracted PS-II particles which contain 4–5 Mn/PS II. Conditions which result in the loss of Mn and O₂ evolution activity from functional membranes, such as incubation in 1.5 mM NH₂OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 ± 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered at g = 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45–55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two ⁵⁵Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn₂(III,III) site or an antiferromagnetically coupled mixed-valence Mn₂(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited spin states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH₂OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein. The intrinsically unstable Mn₂(II,III) oxidation state observed in model compounds favors the assignment of the stable protein oxidation state to the Mn₂(III,III) formulation. This protein exhibits characteristics consistent with an identification with the long-sought Mn site for photosynthetic O₂ evolution. An EPR spectrum having qualitatively similar features is observable in dark-adapted intact, photosynthetic membranes (Dismukes, G.C., Abramowicz, D.A., Ferris, F.K., Mathur, P., Upadrashta, B. and Watnick, P. (1983) in The Oxygen-Evolving System of Plant Photosynthesis (Inoue, Y., ed.), pp. 145–158, Academic Press, Tokyo) and in detergent-extracted, O₂-evolving Photosystem-II particles (Abramowicz, D.A., Raab, T.K. and Dismukes, G.C. (1984) Proceedings of the Sixth International Congress on Photosynthesis (Sybesma, C., ed.), Vol. I, pp. 349–354, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands), thus establishing a direct link with the O₂ evolving complex.     

Mono-manganese mechanism of the photosytem II water splitting reaction by a unique Mn4Ca cluster

The molecular mechanism of the water oxidation reaction in photosystem II (PSII) of green plants remains a great mystery in life science. This reaction is known to take place in the oxygen evolving complex (OEC) incorporating four manganese, one calcium and one chloride cofactors, that is light-driven to cycle four intermediates, designated S₀ through S₄, to produce four protons, five electrons and lastly one molecular oxygen, for indispensable resources in biosphere. Recent advancements of X-ray crystallography models established the existence of a catalytic Mn₄Ca cluster ligated by seven protein amino acids, but its functional structure is not yet resolved. The ¹⁸O exchange rates of two substrate water molecules were recently measured for four S_i-state samples (i = 0-3) leading to ³⁴O₂ and ³⁶O₂ formations, revealing asymmetric substrate binding sites significantly depending on the S_i-state. In this paper, we present a chemically complete model for the Mn₄Ca cluster and its surrounding enzyme field, which we found out from some possible models by using the hybrid density functional theoretic geometry optimization method to confirm good agreements with the 3.0 Å resolution PSII model [B. Loll, J. Kern, W. Saenger, A. Zouni , J. Biesiadka, Nature 438 (2005) 1040-1044] and the S-state dependence of ¹⁸O exchange rates [W. Hillier and T. Wydrzynski, Phys. Chem. Chem. Phys. 6 (2004) 4882-4889]. Furthermore, we have verified that two substrate water molecules are bound to asymmetric cis-positions on the terminal Mn ion being triply bridged (μ-oxo, μ-carboxylato, and a hydrogen bond) to the Mn₃CaO₃(OH) core, by developing a generalized theory of ¹⁸O exchange kinetics in OEC to obtain an experimental evidence for the cross exchange pathway from the slow to the fast exchange process. Some important experimental data will be discussed in terms of this model and its possible tautomers, to suggest that a cofactor, Cl⁻ ion, may be bound to CP43-Arg357 nearby Ca²⁺ ion and that D1-His337 may be used to trap a released proton only in the S₂-state.     

The two forms of the S2 state multiline signal in Photosystem II: effect of methanol and ethanol

The characteristic Mn hyperfine ‘multiline’ signal exhibited in the S₂ state of the oxygen-evolving complex (OEC) complex of Photosystem II (PSII) has been shown to be heterogeneous in character. In this study, we have explored the effects that influence the proportions of the two forms of the S₂ state multiline signal present in any sample. The narrow form of the signal is lost upon storage (weeks) at 77 K, whereas the broad form remains. In particular, we explore the roles of ethanol and methanol as well as effects of the second turnover of the enzyme on storage of the sample at 77 K. We find that in samples containing methanol, the narrow form may predominate upon the first flash, but the broad form predominates on the fifth flash and also in samples containing ethanol.     

Formation and flash-dependent oscillation of the S2-state multiline EPR signal in an oxygen-evolving Photosystem-II preparation lacking the three extrinsic proteins in the oxygen-evolving system

In PS-II-enriched membranes lacking the three extrinsic water-soluble proteins in the oxygen-evolving system (18, 24 and 33 kDa), but still evolving oxygen to some extent, the formation of the multiline EPR signal originating from the S₂-state is dependent on the concentration of Cl⁻. In 200 mM Cl⁻ the multiline signal was observed after the first flash and oscillated with the flash number with a period of four. At 20 mM Cl⁻ no signal could be observed in this material. These results suggest that the extrinsic proteins are not necessary for multiline signal formation and that complete advancement through the S-states can occur in their absence when sufficient Cl⁻ is present.     

The state of manganese in the photosynthetic apparatus: 5. The chloride effect in photosynthetic oxygen evolution. Is halide coordinated to the EPR-active manganese in the O2-evolving complex? Studies of the substructure of the low-temperature multiline EPR signal

The role of chloride in the manganese-containing oxygen-evolving complex of Photosystem II has been studied by observing the amplitude of the multiline EPR signal as a function of Cl⁻ concentration or when Cl⁻ is replaced by Br⁻ or F⁻. The correlation of the multiline EPR signal intensity and O₂ activity with the concentration of Cl⁻ shows that chloride is involved in oxygen evolution at the S₂ or earlier S states, and that it is necessary for the production of an EPR-detectable S₂ state. We have developed a new method for the preparation of subchloroplast PS II particles containing Br⁻ and F⁻) and have used these particles for studying the EPR fine structure at high resolution. The fine structure shows a multiplet of 4–6 lines with 10–15 G spacing; at the resolution of our experiment there are no significant differences between the Cl⁻-and Br⁻-containing samples, suggesting that the halide is not a ligand of the EPR-active Mn. Various structural possibilities for the Mn complex, which would account for the observed fine structure of the multiline EPR spectrum are discussed.     

1H NMR studies of the Fe7S8 ferredoxin from Bacillus schlegelii: a further attempt to understand Fe3S4 clusters

 The oxidized Fe₇S₈ ferredoxin from Bacillus schlegelii, containing both [Fe₃S₄]⁺ and [Fe₄S₄]²⁺ clusters, has been investigated by ¹H NMR spectroscopy. An extensive sequence-specific assignment of the hyperfine-shifted resonances has been obtained by making use of a computer-generated structural model. The pattern and the temperature dependence of the hyperfine shifts of the β-CH₂ protons of the cysteines coordinating the [Fe₃S₄]⁺ cluster are rationalized in terms of magnetic interactions between the iron ions. The same approach holds for the hyperfine coupling with ⁵⁷Fe. It is shown that the magnetic interactions are more asymmetric in Fe₇S₈ ferredoxins than in Fe₃S₄ ferredoxins. The NMR non-observability of the β-CH₂ protons of coordinated cysteines in the one-electron-reduced form has been discussed. Received: 19 June 1996 / Accepted: 2 August 1996     

Evidence for an association between a 33 kDa extrinsic membrane protein,manganese and photosynthetic oxygen evolution. I. Correlation with the S2 multiline EPR signal

The removal of peripheral membrane proteins of a molecular mass of 17 and 23 kDa by washing of spinach Photosystem-II (PS II) membranes in 1 M salt between pH 4.5 and 6.5 produces a minimal loss of the S₁ → S₂ reaction, as seen by the multiline EPR signal for the S₂ state of the water-oxidizing complex, while reversibly inhibiting O₂ evolution. The multiline EPR signal simplifies from a ‘19-line’ spectrum to a ‘16-line’ spectrum, suggestive of partial uncoupling of a cluster of 3 or 4 to yield photo-oxidation of a binuclear Mn site. Alkaline salt washing progressively releases a 33 kDa peripheral protein between pH 6.5 and 9.5, in direct parallel with the loss of O₂ evolution and the S₂ multiline EPR signal. The 33 kDa protein can be partially removed (20%) at pH 8.0 prior to managanese release. Salt treatment releases four Mn ions between pH 8.0 and 9.5 with the first 2 or 3 Mn ions released cooperatively. A common binding site is thus suggested in agreement with earlier EPR spectroscopic data establishing a tetranuclear Mn site. At least two of these Mn ions bind directly at a site in the PS II complex for which photooxidation by the reaction center is controlled by the 33 kDa protein. The washing of PS II membranes with 1 M CaCl₂ to affect the release of the 33 kDa protein, while preserving Mn binding to the membrane (Ono, T.-A. and Inoue, Y. (1983) FEBS Lett. 164, 255–260), is found to leave some 33 kDa protein undissociated in proportion to the extent of O₂ evolution and S₂ multiline yield. These depleted membranes do not oxidize water or produce the normal S₂ state without the binding of the 33 kDa protein. A method for the accurate determination of relative concentrations of the peripheral membrane proteins using gel electrophoresis is presented.     

Characterization of the active site and insight into the binding mode of the anti-angiogenesis agent fumagillin to the manganese(II)-loaded methionyl aminopeptidase from Escherichia coli

EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (~2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as [MnMn(EcMetAP-I)]). The spectra for each sample were indistinguishable except that the spectrum of [MnMn(EcMetAP-I)] was twice as intense. The EPR spectrum of [MnMn(EcMetAP-I)] exhibited the characteristic six-line g2 EPR signal of mononuclear Mn(II) with A_av(⁵⁵Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence. This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen atoms. Other features in the spectrum were observed only as the temperature was raised from that of liquid helium. The temperature dependences of these features are consistent with their assignment to excited state transitions in the S=1, 2 ... 5 non-Kramers doublets, due to two antiferromagnetically coupled Mn(II) ions with an S=0 ground state. This assignment is supported by the observation of a characteristic 4.5 mT hyperfine pattern, and by the presence of signals in the parallel mode consistent with a non-Kramers spin ladder. Upon the addition of the anti-angiogenesis agent fumagillin to [MnMn(EcMetAP-I)], very small changes were observed in the EPR spectrum. MALDI-TOF mass spectrometry indicated that fumagillin was, however, covalently coordinated to EcMetAP-I. Therefore, the inhibitory action of this anti-angiogenesis agent on EcMetAP-I appears to involve covalent binding to a polypeptide component at or near the active site rather than direct binding to the metal ions.     

The state of manganese in the photosynthetic apparatus: 4. Structure of the manganese complex in Photosystem II studied using EXAFS spectroscopy. The S1 state of the O2-evolving Photosystem II complex from spinach

The structure of the Mn complex in the oxygen-evolving system and its mechanistic relation to photosynthetic oxygen evolution are poorly understood, though many studies have established that membrane-bound Mn plays an active role. Recently established procedures for isolating oxygen-evolving subchloroplast Photosystem II (PS II) preparations and the discovery of a light-induced multiline EPR signal attributable to the S₂ state of the O₂-evolving complex have facilitated the preparation of samples well characterized in the S₁ and S₂ states. We have used extended X-ray absorption fine structure (EXAFS) spectroscopy to probe the ligand environment of Mn in PS II particles from spinach, and in this report we present our results. The essential feature of the EXAFS results are that at least two Mn atoms per PS II reaction center occur as a binuclear species with a metal-metal distance of approx. 2.7 Å, with low Z atoms, N or O, at a distance of approx. 1.75 Å and at approx. 1.98 Å, which are characteristic of bridging and terminal ligands. These results agree well with those derived from whole chloroplasts that provided the first evidence for a binuclear manganese complex (Kirby, J.A., Robertson, A.S., Smith, J.P., Thompson, A.C., Cooper, S.R. and Klein, M.P. (1981) J. Am. Chem. Soc. 103, 5529–5537).