Multiline Electron Paramagnetic Resonance Research Articles

Insights into the protonation state and spin structure for the g = 2 multiline electron paramagnetic resonance signal of the oxygen-evolving complex.

In photosystem II (PSII), one-electron oxidation of the most stable oxidation state of the Mn4CaO5 cluster (S1) leads to formation of two distinct states, the open-cubane S2 conformation [Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV)] with low spin and the closed-cubane S2 conformation [Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III)] with high spin. In electron paramagnetic resonance (EPR) spectroscopy, the open-cubane S2 conformation exhibits a g = 2 multiline signal. However, its protonation state remains unclear. Here, we investigated the protonation state of the open-cubane S2 conformation by calculating exchange couplings in the presence of the PSII protein environment and simulating the pulsed electron-electron double resonance (PELDOR). When a ligand water molecule, which forms an H-bond with D1-Asp61 (W1), is deprotonated at dangling Mn4(IV), the first-exited energy (34 cm-1) in manifold spin excited states aligns with the observed value in temperature-dependent pulsed EPR analyses, and the PELDOR signal is best reproduced. Consequently, the g = 2 multiline signal observed in EPR corresponds to the open-cubane S2 conformation with the deprotonated W1 (OH-).

Alteration of the O2-Producing Mn4Ca Cluster in Photosystem II by the Mutation of a Metal Ligand.

The O2-evolving Mn4Ca cluster in photosystem II (PSII) is arranged as a distorted Mn3Ca cube that is linked to a fourth Mn ion (denoted as Mn4) by two oxo bridges. The Mn4 and Ca ions are bridged by residue D1-D170. This is also the only residue known to participate in the high-affinity Mn(II) site that participates in the light-driven assembly of the Mn4Ca cluster. In this study, we use Fourier transform infrared difference spectroscopy to characterize the impact of the D1-D170E mutation. On the basis of analyses of carboxylate and carbonyl stretching modes and the O-H stretching modes of hydrogen-bonded water molecules, we show that this mutation alters the extensive network of hydrogen bonds that surrounds the Mn4Ca cluster in the same manner as that of many other mutations. It also alters the equilibrium between conformers of the Mn4Ca cluster in the dark-stable S1 state so that a high-spin form of the S2 state is produced during the S1-to-S2 transition instead of the low-spin form that gives rise to the S2 state multiline electron paramagnetic resonance signal. The mutation may also change the coordination mode of the carboxylate group at position 170 to unidentate ligation of Mn4. This is the first mutation of a metal ligand in PSII that substantially impacts the spectroscopic signatures of the Mn4Ca cluster without substantially eliminating O2 evolution. The results have significant implications for our understanding of the roles of alternate active/inactive conformers of the Mn4Ca cluster in the mechanism of O2 formation.

Assessment of the manganese cluster’s oxidation state via photoactivation of photosystem II microcrystals

Knowledge of the manganese oxidation states of the oxygen-evolving Mn4CaO5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S2 state of this cluster. This signal implies an overall oxidation state of either Mn(III)3Mn(IV) or Mn(III)Mn(IV)3 for the S2 state. These 2 competing assignments are commonly known as "low oxidation (LO)" and "high oxidation (HO)" models of the Mn4CaO5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn4CaO5 cluster starting from Mn(II) and Mn-free PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn4CaO5 cluster's oxidation states.

Energetics of the S2 State Spin Isomers of the Oxygen-Evolving Complex of Photosystem II.

The S2 redox intermediate of the oxygen-evolving complex in photosystem II is present as two spin isomers. The S = 1/2 isomer gives rise to a multiline electron paramagnetic resonance (EPR) signal at g = 2.0, whereas the S = 5/2 isomer exhibits a broad EPR signal at g = 4.1. The electronic structures of these isomers are known, but their role in the catalytic cycle of water oxidation remains unclear. We show that formation of the S = 1/2 state from the S = 5/2 state is exergonic at temperatures above 160 K. However, the S = 1/2 isomer decays to S1 more slowly than the S = 5/2 isomer. These differences support the hypotheses that the S3 state is formed via the S2 state S = 5/2 isomer and that the stabilized S2 state S = 1/2 isomer plays a role in minimizing S2QA- decay under light-limiting conditions.

Dominant Stable Radicals in Irradiated Sucrose: g Tensors and Contribution to the Powder Electron Paramagnetic Resonance Spectrum

Ionizing radiation induces a composite, multiline electron paramagnetic resonance (EPR) spectrum in sucrose, that is stable at room temperature and whose intensity is indicative of the radiation dose. Recently, the three radicals which dominate this spectrum were identified and their proton hyperfine tensors were accurately determined. Understanding the powder EPR spectrum of irradiated sucrose, however, also requires an accurate knowledge of the g tensors of these radicals. We extracted these tensors from angular dependent electron nuclear double resonance-induced EPR measurements at 110 K and 34 GHz. Powder spectrum simulations using this completed set of spin Hamiltonian parameters are in good agreement with experimentally recorded spectra in a wide temperature and frequency range. However, as-yet nonidentified radicals also contribute to the EPR spectra of irradiated sucrose in a non-negligible way.

The reduced S<sub>-1</sub> and S<sub>-2</sub> oxidation states of the O<sup>2</sup>-evolving complex of photosystem II: An EPR microwave power saturation study

Progressive microwave power saturation (P1/2) measurements have been performed on the tyrosine D radical (YD •) of photosystem II (PSII) in order to examine its relaxation enhancement by the oxygen-evolving complex (OEC) poised to the reduced S−1 and S−2 oxidation states by NO treatment. Analysis of the power saturation curves showed that the S−1 oxidation state of the OEC does not enhance the relaxation of YD •: it therefore possesses a diamagnetic ground state. In contrast, the Mn(II)-Mn(III) multiline electron paramagnetic resonance (EPR) signal characteristic of the S−2 oxidation state of the OEC was shown to provide a relaxation enhancement pathway for YD •, however less efficient relative to the one provided by the S2-state multiline EPR signal. We also examined the YD • relaxation enhancement characteristics of the EPR-silent oxidation state produced after brief (1–5 min) dark incubation at 0°C of a PSII sample poised to the EPRactive S−2 state. This EPR-silent oxidation state denoted as “0°C incubation” state was shown to possess remarkably similar P1/2 values with the EPR-active S−2 state in the overall examined temperature range (6–20 K). In addition, these values remained unchanged after successive cycles of the OEC between the EPR-active S−2 state and the “0°C incubation” state. The data presented in this work point to the conclusion that the “0°C incubation” state is indeed an S−2 oxidation state with half-integer spin.

Glutamate-354 of the CP43 polypeptide interacts with the oxygen-evolving Mn4Ca cluster of photosystem II: a preliminary characterization of the Glu354Gln mutant

In the recent X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is assigned as a ligand of the O2-evolving Mn4Ca cluster. In this communication, a preliminary characterization of the CP43-Glu354Gln mutant of the cyanobacterium Synechocystis sp. PCC 6803 is presented. The steady-state rate of O2 evolution in the mutant cells is only approximately 20% compared with the wild-type, but the kinetics of O2 release are essentially unchanged and the O2-flash yields show normal period-four oscillations, albeit with lower overall intensity. Purified PSII particles exhibit an essentially normal S2 state multiline electron paramagnetic resonance (EPR) signal, but exhibit a substantially altered S2-minus-S1 Fourier transform infrared (FTIR) difference spectrum. The intensities of the mutant EPR and FTIR difference spectra (above 75% compared with wild-type) are much greater than the O2 signals and suggest that CP43-Glu354Gln PSII reaction centres are heterogeneous, with a minority fraction able to evolve O2 with normal O2 release kinetics and a majority fraction unable to advance beyond the S2 or S3 states. The S2-minus-S1 FTIR difference spectrum of CP43-Glu354Gln PSII particles is altered in both the symmetric and asymmetric carboxylate stretching regions, implying either that CP43-Glu354 is exquisitely sensitive to the increased charge that develops on the Mn4Ca cluster during the S1-->S2 transition or that the CP43-Glu354Gln mutation changes the distribution of Mn(III) and Mn(IV) oxidation states within the Mn4Ca cluster in the S1 and/or S2 states.

Probing the Functional Role of Ca2+ in the Oxygen-Evolving Complex of Photosystem II by Metal Ion Inhibition

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn4), a redox-active tyrosine residue (YZ), and Ca2+ and Cl- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O2. Ca2+ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca2+ has been revealed to be associated with the Mn4 cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca2+ in oxygen evolution remains unclear. In this study, we probe the role of Ca2+ in oxygen evolution by monitoring the S1 to S2 state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy3+, Cu2+, and Cd2+ ions. By using a cation-exchange procedure in which Ca2+ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy3+, Cu2+, and Cd2+ ions. EPR spectroscopy is used to quantitate the number of bound Dy3+ and Cu2+ ions per PSII center and to determine the proximity of Dy3+ to other paramagnetic centers in PSII. We observe, for the first time, the S2 state multiline electron paramagnetic resonance (EPR) signal in Dy3+- and Cd2+-inhibited PSII and conclude that the Ca2+ cofactor is not specifically required for the S1 to S2 state transition of PSII. This observation provides direct support for the proposal that Ca2+ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca2+ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca2+ and Sr2+, which have similar Lewis acidities.

Evidence for PSII donor-side damage and photoinhibition induced by cadmium treatment on rice ( Oryza sativa L.)

The effects of cadmium (from 7.5 to 75 μM) on chloroplasts of rice were studied at the structural and biochemical level. Loss of pigments, reduction of thylakoids and decrease in oxygen evolution and Fv/Fm ratio occur in leaves following cadmium treatment. However, the amount of photosystem II reaction center proteins and that of its light harvesting complex is not affected, indicating that cadmium does not adversely influence the structural organization of this photosystem. In thylakoids isolated from cadmium-treated plants a loss in the capability to reduce 2,6-dichlorophenolindophenol is observed, which is partially restored if diphenylcarbazide is used as an electron donor, indicating that cadmium affects water splitting activity. In thylakoids isolated from control plants and treated with cadmium, diphenylcarbazide preserves most of the photosystem II activity lost after incubation with cadmium; most of the S 2 multiline electron paramagnetic resonance signal from the manganese cluster is lost, whereas the TyrD + and other signals are retained. Light-induced photosystem II damage, in vitro, is promoted by Cd-treatment as deduced from the mobility shift of the D1 protein observed by immunoblot.

Photosensitized Oxidation and Inactivation of Pyocyanin, a Virulence Factor of Pseudomonas aeruginosa

Pyocyanin (PyO-) (1-hydroxy-5-methylphenazine) is a cytotoxic compound secreted by Pseudomonas aeruginosa, an omnipresent bacterium and a human pathogen. We report that visible light illumination in the presence of rose bengal, or riboflavin, in aerated solutions (pH 7.0-7.2) induces irreversible loss of the pigment's characteristic absorption band at 690 nm, indicating its oxidation. This photobleaching was paralleled by generation of a multiline Electron Paramagnetic Resonance (EPR) spectrum attributed to a PyO(-)-derived radical. The reaction was dependent on the presence of air, sensitizers and light, was inhibited by sodium azide and was unaffected by ethanol. This suggests that PyO- was oxidized largely via singlet oxygen and that hydroxyl radicals were not involved. The photochemically modified pigment was less efficient in oxidizing NAD(P)H and generated less superoxide (by approximately 50%) than the intact PyO-, indicating its partial inactivation. 1-Methoxy-5-methylphenazine, a PyO- analog in which the -O- moiety was replaced by the methoxy group (-OMe), was resistant to oxidation, suggesting that oxidation of PyO- involves its phenolate moiety. These results also suggest that photosensitization could be a potentially useful method for inactivation of PyO- and, possibly, detoxification of superficial wounds (skin, eye) infected with P. aeruginosa.

Ca 2+ Function in Photosynthetic Oxygen Evolution Studied by Alkali Metal Cations Substitution

Effects of adding monovalent alkali metal cations to Ca 2+-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca 2+-dependent oxygen evolution was competitively inhibited by K +, Rb +, and Cs +, the ionic radii of which are larger than the radius of Ca 2+ but not inhibited significantly by Li + and Na +, the ionic radii of which are smaller than that of Ca 2+. Ca 2+-depleted membranes without metal cation supplementation showed normal S 2 multiline electron paramagnetic resonance (EPR) signal and an S 2Q A − thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li + or Na + showed properties similar to those of the Ca 2+-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K +, Rb +, or Cs + was elevated to ∼38°C which coincided with that of Y D +Q A − TL band, and no S 2 EPR signals were detected. The K +-induced high-temperature TL band and the S 2Q A − TL band were interconvertible by the addition of K + or Ca 2+ in the dark. Both the Ca 2+-depleted and the K +-substituted membranes showed the narrow EPR signal corresponding to the S 2Y Z + state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca 2+-binding site crucially affect the properties of the manganese cluster.

Comparative studies of the S 0 and S 2 multiline electron paramagnetic resonance signals from the manganese cluster in Photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is one of the major techniques used to analyse the structure and function of the water oxidising complex (WOC) in Photosystem II. The discovery of an EPR signal from the S 0 state has opened the way for new experiments, aiming to characterise the S 0 state and elucidate the differences between the different S states. We present a review of the biochemical and biophysical characterisation of the S 0 state multiline signal that has evolved since its discovery, and compare these results to previous and recent data from the S 2 multiline signal. We also present some new data from the S 2 state reached on the second turnover of the enzyme.

Proton equilibria in the manganese cluster of photosystem II control the intensities of the S(0) and S(2) state g approximately 2 electron paramagnetic resonance signals.

We have studied the pH effect on the S(0) and S(2) multiline electron paramagnetic resonance (EPR) signals from the water-oxidizing complex of photosystem II. Around pH 6, the maximum signal intensities were detected. On both the acidic and alkaline sides of pH 6, the intensities of the EPR signals decreased. Two pKs were determined for the S(0) multiline signal; pK(1) = 4.2 +/- 0.2 and pK(2) = 8.0 +/- 0.1, and for the S(2) multiline signal the pKs were pK(1) = 4.5 +/- 0.1 and pK(2) = 7.6 +/- 0.1. The intensity of the S(0)-state EPR signal was partly restored when the pH was changed from acidic or alkaline pH back to pH approximately 6. In the S(2) state we observed partial recovery of the multiline signal when going from alkaline pH back to pH approximately 6, whereas no significant recovery of the S(2) multiline signal was observed when the pH was changed from acidic pH back to pH approximately 6. Several possible explanations for the intensity changes as a function of pH are discussed. Some are ruled out, such as disintegration of the Mn cluster or decay of the S states and formal Cl(-) and Ca(2+) depletion. The altered EPR signal intensities probably reflect the protonation/deprotonation of ligands to the Mn cluster or the oxo bridges between the Mn ions. Also, the possibility of decreased multiline signal intensities at alkaline pH as an effect of changed redox potential of Y(Z) is put forward.

Investigation of the interaction of the water oxidising manganese complex of photosystem II with the aqueous solvent environment

Interaction of the water oxidising manganese complex of photosystem II with the aqueous environment has been investigated using electron paramagnetic resonance spectroscopy and electron spin echo envelope modulation spectroscopy to detect interaction of [ 2H]methanol with the complex in the S 2 state. The experiments show that the classical S 2 multiline signal is associated with a manganese environment which is not exposed to the aqueous medium. An electron paramagnetic resonance spectroscopy signal, also induced by 200 K illumination, showing 2H modulation by methanol in the medium and a modified multiline electron paramagnetic resonance spectroscopy signal formed in parallel to it, are suggested to be associated with a second manganese environment exposed to the medium.

Simulation of S2-State Multiline EPR Signal in Oriented Photosystem II Membranes: Structural Implications for the Manganese Cluster in an Oxygen-Evolving Complex

High-quality angular-dependent spectra of multiline electron paramagnetic resonance (EPR) signals from the S2-state Mn cluster in a photosynthetic oxygen-evolving complex (OEC) were obtained for partially oriented photosystem (PS) II membranes, and the magnetic structure of the Mn cluster has been studied by simulation analysis. The angular-dependent multiline spectra were simulated by taking into account the anisotropic properties of both hyperfine tensors of intrinsic Mn ions and g-tensor of the cluster in a tetranuclear model. The best-fit parameters for the simulation indicate that (a) the oxidation state of the S2-state Mn cluster is Mn(III, IV, IV, IV), (b) the electronic orbital configuration of the Mn(III) ion is (dpi)3[dz2(sigma))]1, (c) the effective g-tensor of the Mn cluster and the hyperfine tensor of the Mn(III) ion are axially symmetric, and their principal z-axes are nearly collinear each other, and (d) the z-axis of the dz2 orbital of the Mn(III) ion and the normal of the thylakoid membrane are at an angle of 50.1 +/- 1.8 degrees. The results are compatible with those of the oriented XAFS study [Mukerji, I., et al. (1994) Biochemistry 33, 9712-9721], and indicate that the O-O vector of the putative di-mu-oxo bridged Mn(III)-Mn(IV) dimer unit in the Mn cluster tilts by 43-56 degrees with respect to the normal of thylakoid membrane. A model of the arrangement of the di-mu-oxo bridged Mn(III)-Mn(IV) unit with respect to the thylakoid membrane is proposed.

Simulation of the S2 state multiline electron paramagnetic resonance signal of photosystem II: a multifrequency approach

The S2 state electron paramagnetic resonance (EPR) multiline signal of Photosystem II has been simulated at Q-band (35 Ghz), X-band (9 GHz) and S-band (4 GHz) frequencies. The model used for the simulation assumes that the signal arises from an essentially magnetically isolated MnIII-MnIV dimer, with a ground state electronic spin ST = 1/2. The spectra are generated from exact numerical solution of a general spin Hamiltonian containing anisotropic hyperfine and quadrupolar interactions at both Mn nuclei. The features that distinguish the multiline from the EPR spectra of model manganese dimer complexes (additional width of the spectrum (195 mT), additional peaks (22), internal "superhyperfine" structure) are plausibly explained assuming an unusual ligand geometry at both Mn nuclei, giving rise to normally forbidden transitions from quadrupole interactions as well as hyperfine anisotropy. The fitted parameters indicate that the hyperfine and quadrupole interactions arise from Mn ions in low symmetry environments, corresponding approximately to the removal of one ligand from an octahedral geometry in both cases. For a quadrupole interaction of the magnitude indicated here to be present, the MnIII ion must be 5-coordinate and the MnIV 5-coordinate or possibly have a sixth, weakly bound ligand. The hyperfine parameters indicate a quasi-axial anisotropy at MnIII, which while consistent with Jahn-Teller distortion as expected for a d4 ion, corresponds here to the unpaired spin being in the ligand deficient, z direction of the molecular reference axis. The fitted parameters for MnIV are very unusual, showing a high degree of anisotropy not expected in a d3 ion. This degree of anisotropy could be qualitatively accounted for by a histidine ligand providing pi backbonding into the metal dxy orbital, together with a weakly bound or absent ligand in the x direction.

Resolution of the paradox of ammonia and hydroxylamine as substrate analogs for the water-oxidation reaction catalyzed by photosystem II

The O/sub 2/-evolving center of photosystem II (PSII) contains a Mn tetramer complex that serves as the catalyst for the H/sub 2/O-oxidation reaction. In a ligand-substitution reaction thought to be analogous to the substrate-binding reaction, NH/sub 3/ coordinates to the Mn complex only after formation of the S/sub 2/ state, which then exhibits an altered S/sub 2/-state multiline electron paramagnetic resonance (EPR) signal. The free-base form of hydroxylamine binds to a Cl/sup -/-binding site prior to reacting with the Mn complex; subsequently, hydroxylamine probably reduces the Mn complex from the S/sub 1/ state to the S..sqrt../sub 1/ state through an outer-sphere electron-transfer mechanism. The rate of hydroxylamine's reaction with the Mn complex is reduced about 17-fold with each added N-methyl substituent, showing that the binding site is sterically selective for small ligands. NH/sub 3/ also binds to the O/sub 2/-evolving center in the S/sub 1/ state in a manner inversely dependent on the Cl/sup -/ concentration, causing the Mn complex to exhibit a g = 4.1 EPR signal in lieu of the multiline EPR signal in the S/sub 2/ state. The results suggest that primary amines and hydroxylamines bind to the O/sub 2/-evolving center in the S/sub 1/ state atmore » the same Cl/sup -/-binding site. Thus, two ligand-binding sites exist in the O/sub 2/-evolving center. One site is located on the Mn complex and is restricted to binding NH/sub 3/ and presumably H/sub 2/O. This site is assigned to the substrate-binding site. A second site is identified as the Cl/sup -/-binding site; primary amines and hydroxylamines compete with Cl/sup -/ for coordination to this site. In view of the finding that hydroxylamines bind to the Cl/sup -/-binding site, they conclude that hydroxylamines should not be considered as substrate analogues for the H/sub 2/O-oxidation reaction catalyzed by PSII.« less

Comparison of the structure of the manganese complex in the S1 and S2 states of the photosynthetic oxygen-evolving complex: an x-ray absorption spectroscopy study

A Mn-containing enzyme complex is involved in the oxidation of H2O to O2 in algae and higher plants. X-ray absorption spectroscopy is well suited for studying the structure and function of Mn in this enzyme complex. Results of X-ray K-edge and extended X-ray absorption fine structure (EXAFS) studies of Mn in the S1 and S2 states of the photosynthetic O2-evolving complex in photosystem II preparations from spinach are presented in this paper. The S2 state was prepared by illumination at 190 K or by illumination at 277 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); these are protocols that limit the photosystem II reaction center to one turnover. Both methods produce an S2 state characterized by a multiline electron paramagnetic resonance (EPR) signal. An additional protocol, illumination at 140 K, produces as a state characterized by the g = 4.1 EPR signal. We have previously observed a shift to higher energy in the X-ray absorption K-edge energy of Mn upon advancement from the dark-adapted S1 state to the S2 state produced by illumination at 190 K [Goodin, D. B., Yachandra, V. K., Britt, R. D., Sauer, K., & Klein, M. P. (1984) Biochim. Biophys. Acta 767, 209-216]. The Mn K-edge spectrum of the 277 K illuminated sample is similar to that produced at 190 K, indicating that the S2 state is similar when produced at 190 or 277 K.(ABSTRACT TRUNCATED AT 250 WORDS)