Alain Boussac

Detection of the water-binding sites of the oxygen-evolving complex of Photosystem II using W-band 17O electron-electron double resonance-detected NMR spectroscopy.

Water binding to the Mn(4)O(5)Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S(2) state was studied via H(2)(17)O- and (2)H(2)O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating (17)O (I = 5/2) nuclei were detected using W-band (94 GHz) electron-electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal (15)N (I = ½) labeling was employed to clearly discriminate the (17)O hyperfine couplings that overlap with (14)N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of (17)O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH(2) ligand; and (iii) Mn/Ca-H(2)O ligand(s). These assignments are based on (17)O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH(3) perturbation of the (17)O signal envelope and density functional theory calculations. The relative orientation of the putative (17)O μ-oxo bridge hyperfine tensor to the (14)N((15)N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn(3)O(3)Ca open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All (17)O signals were seen even after very short (≤15 s) incubations in H(2)(17)O suggesting that all exchange sites identified could represent bound substrate in the S(1) state including the μ-oxo bridge. (1)H/(2)H (I = ½, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small (1)H/(2)H couplings observed require that all the μ-oxo bridges of the Mn(4)O(5)Ca cluster are deprotonated in the S(2) state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S(4).

Structural changes in the Mn4Ca cluster and the mechanism of photosynthetic water splitting

Photosynthetic water oxidation, where water is oxidized to dioxygen, is a fundamental chemical reaction that sustains the biosphere. This reaction is catalyzed by a Mn4Ca complex in the photosystem II (PS II) oxygen-evolving complex (OEC): a multiprotein assembly embedded in the thylakoid membranes of green plants, cyanobacteria, and algae. The mechanism of photosynthetic water oxidation by the Mn4Ca cluster in photosystem II is the subject of much debate, although lacking structural characterization of the catalytic intermediates. Biosynthetically exchanged Ca/Sr-PS II preparations and x-ray spectroscopy, including extended x-ray absorption fine structure (EXAFS), allowed us to monitor Mn–Mn and Ca(Sr)–Mn distances in the four intermediate S states, S0 through S3, of the catalytic cycle that couples the one-electron photochemistry occurring at the PS II reaction center with the four-electron water-oxidation chemistry taking place at the Mn4Ca(Sr) cluster. We have detected significant changes in the structure of the complex, especially in the Mn–Mn and Ca(Sr)–Mn distances, on the S2-to-S3 and S3-to-S0 transitions. These results implicate the involvement of at least one common bridging oxygen atom between the Mn–Mn and Mn–Ca(Sr) atoms in the O–O bond formation. Because PS II cannot advance beyond the S2 state in preparations that lack Ca(Sr), these results show that Ca(Sr) is one of the critical components in the mechanism of the enzyme. The results also show that Ca is not just a spectator atom involved in providing a structural framework, but is actively involved in the mechanism of water oxidation and represents a rare example of a catalytically active Ca cofactor.

X-ray crystallography identifies two chloride binding sites in the oxygen evolving centre of Photosystem II

Bromide anomalous X-ray diffraction analyses have been used to locate chloride binding sites in the vicinity of the water splitting/oxygen evolving centre (OEC) of Photosystem II. Three-dimensional crystals of PSII from Thermosynechococcus elongatus were grown from (i) isolated PSII crystals infiltrated with bromide or (ii) PSII obtained from cells cultured in a medium in which the chloride content was totally replaced by bromide. In either case, the anomalous diffraction yielded the same result, the existence of two bromide binding sites in the vicinity of the OEC. Neither are in the first coordination sphere of the Mn and Ca ions which form the catalytic centre of the OEC, being about 6 to 7 A from the metal-cluster. Site 1 is located close to the side chain nitrogen of D2-K317 and the backbone nitrogen of D1-Glu333 while Site 2 is adjacent to backbone nitrogens of CP43-Glu354 and D1-Asn338. Their positioning close to postulated hydrophilic channels may suggest a role in proton removal from, or substrate access to, the OEC.

Ammonia binding to the oxygen-evolving complex of photosystem II identifies the solvent-exchangeable oxygen bridge (μ-oxo) of the manganese tetramer

The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn4O5Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn4O5Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522–3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that (i) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55–60]}; (ii) ammonia displaces a water ligand to the outer manganese (MnA4-W1); and (iii) as W1 is trans to O5, ammonia binding elongates the MnA4-O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water.

Effect of the 33-kDa protein on the S-state transitions in photosynthetic oxygen evolution

The effect of the extrinsic 33-kDa protein on the photosynthetic oxygen evolution was studied by comparing spinach Photosystem II particles depleted of the 33-kDa protein with those reconstituted with the protein. The light-intensity dependence of the oxygen-evolution activity under continuous illumination suggests that a dark step, but not a light step, in the oxygen-evolving reaction is accelerated by the 33-kDa protein. Consistently, the pattern of oxygen yield with a series of short saturating flashes, which showed a maximum on the third flash and a damped oscillation with a period of 4, was not much affected by the removal and rebinding of the 33-kDa protein, when the dark interval between the flashes was long enough, i.e., longer than 0.5 s. The millisecond kinetics of oxygen release after the third flash was retarded by the removal of the 33-kDa protein and stimulated by its rebinding, suggesting that the transition from S3 to S0 is accelerated by the 33-kDa protein. The stability of the S2 and S3 states in darkness was higher in the absence of the 33-kDa protein than its presence.

The origin of 40-50°C thermoluminescence bands in Photosystem II

Abstract We have used thermoluminescence (TL) and EPR measurements of Photosystem II (PS II) from spinach in order to identify charge pairs responsible for TL bands in the region of 40–50°C including the ‘C-band’ (peak V) and the TL band from PS II depleted of calcium. In intact PS II membrane preparations, in the presence of DCMU, a TL band at 50°C is induced following illumination at 77 K. This band decays, at 30°C, with a half-time of 10 min. This decay corresponds to the disappearance of the EPR signal arising from QA− and an accelerated decay of the organic free radical Tyr D+. It is concluded that recombination of this charge pair is probably responsible for the thermoluminescence emission. In PS II preparations that have been depleted of calcium using a salt/EGTA wash followed by rebinding of the extrinsic polypeptides, a TL band is produced at around 45–50°C following 198 K illumination. In such samples a stable S2 state of the water-splitting complex is present, giving rise to a modified form of the EPR multiline signal. During incubation at 30°C in the dark this signal decays with a half-time around 20–25 min. This decay is not accelerated by the presence of QA− induced by low-temperature illumination of the sample. In contrast, low-temperature illumination does result in an acceleration in the decay of Tyr D+, indicating that Tyr D+/Q−A recombination is again the dominant origin of thermoluminescence. In PS II depleted of calcium by incubation at pH 4.0, the possibility that TL emission temperature is determined by a change in the mid-point redox potential of QA (Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89–98) was investigated by comparing TL from equivalent samples of control and Ca2+-depleted PS II. It was shown that the emission temperature of the high temperature TL band induced by illumination at 77 K did not differ significantly between control and treated samples, suggesting that, under the conditions used, the potential of QA does not change significantly.

Midpoint potential of signal II (slow) in Tris-washed photosystem-II particles

Abstract In Tris-washed Photosystem-II particles we are able to induce an EPR signal in the dark by addition of an iridium salt (K2IrCl6). This signal is attributed to signal IIs (slow) (D+) and the redox titration gives an Em value of 760 mV for the couple D + D . On the basis of our previous studies on the equilibrium between D+Z and DZ+ (K = 104) (Boussac, A. and Etienne, A.L. (1982) Biochem. Biophys. Res. Commun. 109, 1200–1205), we therefore attribute a value of 1 V for the Em of the Z + Z couple. A second effect of K2IrCl6 is to modify the spectral characteristics of signal II. We conclude that K2IrCl6 is able to change the environment of the species from which signal IIs and signal IIf originate.

Ca2+ binding to the oxygen evolving enzyme varies with the redox state of the Mn cluster

Oxygen evolution by the mangano‐enzyme of photosystem II is inhibited by Ca2+ depletion induced by NaCl washing and restored by Ca2+ addition. The effectiveness of NaCl treatment in inhibiting oxygen evolution in photosystem II was studied after a series of preilluminating flashes. The susceptibility of the enzyme to NaCl treatment varied with the number of preilluminating flashes and this variation showed an oscillation pattern with a period of four. This pattern is characteristic of cycling through the four long‐lived intermediate states in the enzyme cycle (i.e. the states, S0, S1, S2, S3). The relative extent of inhibition corresponding to each of the S states was as follows: S3 > S0≈S2 > S1. From these results it is concluded that Ca2+ binding is dependent on the S states and that Ca2+ probably plays a fundamental role in the mechanism of water splitting. The results also help to explain the conflicting reports of the extent of inhibition induced by NaCl washing and the controversy over which electron transfer step is inhibited by Ca2+ depletion.

FeIII‐Hydroperoxo and Peroxo Complexes with Aminopyridyl Ligands and the Resonance Raman Spectroscopic Identification of the Fe−O and O−O Stretching Modes

Nonheme Fe(III)-hydroperoxo and Fe(III)-peroxo complexes with aminopyridyl-type ligands have been prepared and characterized by UV/Vis, EPR, mass and Resonance Raman (RR) spectroscopy. The Fe(III)(OOH) species are low-spin and exhibit a deep purple color due to the ligand-to-metal charge transfer (LMCT) hand centered at ca. 550 nm. The RR spectra of the Fe(III)(OOH) complexes display two bands at ca. 620 and 800 cm-1 that are assigned to the respective Fe-O and O-O stretching modes on the basis of the characteristic H/D and 16O/18O frequency shifts. Upon deprotonation, Fe(III)(O2) species are obtained which possess a high-spin configuration of nearly axial symmetry and a LMCT transition in the near infrared (ca. 750 nm). The frequencies of the Fe-O and O-O stretching modes at ca. 465 and 820 cm-1, as well as their respective 16O/18O shifts of -16 and -45 cm-1, indicate an ?2 coordination geometry for the Fe(III)(O2) complex.

Factors influencing the formation of modified S2 EPR signal and the S3 EPR signal in Ca2+-depleted photosystem II

NaCl/EGTA‐washing of photosystem II (PS‐II) results in the removal of Ca2+ and the inhibition of oxygen evolution. Two new EPR signals were observed in such samples: a stable and modified S2 multiline signal and an S3 signal [(1989) Biochemistry 28, 8984‐8989]. Here, we report what factors are responsible for the modifications of the S2 signal and the observation of the S3 signal. The following results were obtained, (i) The stable, modified, S2 multiline signal can be induced by the addition of high concentrations of EGTA or citrate to PS‐II membranes which are already inhibited by Ca2+‐depletion. (ii) The carboxylic acids act in the S3‐state, are much less effective in S2 and have no effect in the S1‐state. (iii) The extrinsic polypeptides (17‐ and 23‐kDa) are not required to observe either the modified S2 signal or the S3 signal. However, they do influence the splitting and the lifetime of the S3 signal, and they seem to have a slight influence on the hyperfine pattern of the S2 signal, (iv) The S3 signal can be observed in Ca2+‐depleted PS‐II which does not exhibit the modified multiline signal. Then, it is proposed that formation of histidine radical during the S2 to S3 transition in Ca2+ ‐depleted PS‐II [(1990) Nature 347, 303‐306] also occurs in functional PS‐II.