We report the first parallel polarization EPR signal from the Mn(III) ion formed by photooxidation of Mn(II) bound at the high affinity Mn-binding site of photosystem II (PSII). This species corresponds to the first photoactivation intermediate formed on the pathway to assembly of the water-splitting Mn cluster. The parallel mode EPR spectrum of the photooxidation product of 1.2/1 stoichiometry Mn(II)/Mn-depleted wildtype Synechocystis sp. PCC 6803 PSII particles consists of six well-resolved transitions split by a relatively small 55 Mn hyperfine coupling (44 G). This spectral signature is absent in photooxidized Mn apoPSII complexes prepared from D1-Asp170Glu and D1-Asp170His mutants, providing direct spectral evidence for a role for this specific D1-Asp170 residue in the initial photoactivation chemistry. Temperature-dependence measurements and spectral simulations performed on the Mn(III) parallel mode EPR signal of the wild-type sample give an axial zero-field splitting value of D ≈ -2.5 cm -1 and a rhombic zero-field splitting value of |E| ≈ 0.269 cm -1 . The negative D value for this d 4 ion is indicative of either a 5 B 1g symmetry ground state of an octahedral Mn(III) geometry or a 5 B 1 symmetry ground state of a five-coordinate square-pyramidal Mn(III) geometry. The parallel mode Mn(III) EPR spectrum obtained from the wild-type photooxidized Mn apoPSII complex is contrasted with that obtained from the five-coordinate Mn(III) form of native Mn superoxide dismutase, which has a trigonal-bipyramidal geometry and a 5 A 1 symmetry ground state giving rise to a positive D value and a much larger 55 Mn hyperfine coupling of 100 G. The D1-Asp170His mutant displays a parallel mode EPR spectrum similar to that observed in a Mn(III) model complex. The D1-Asp170Glu mutant shows no parallel mode spectrum, but in perpendicular mode it shows a broad feature near g ) 5 which has spectral characteristics of an S ) 3 / 2 Mn(IV) ion. This suggests that this mutant provides a binding site with a less positive Mn(III)/ Mn(IV) reduction potential.Photosystem II of oxygenic photosynthesis utilizes a tetranuclear Mn cluster and a redox active tyrosine residue (Y Z ) to couple the reduction of the photooxidized Chl species P 680 + to the enzymatic oxidation of water to dioxygen. 1 The same photochemistry is utilized to assemble the Mn cluster through a process termed "photoactivation", 2-6 where bioavailable Mn(II) ions are oxidized to the higher Mn(III)/Mn(IV) valencies of the catalytic cluster. The first photooxidation event occurs at a unique high-affinity Mn(II) binding site, where Mn(II) is oxidized to Mn(III) by the neutral Y Z • radical formed upon the coupled deprotonation and oxidation of Y Z by P 680 + . 3 It is likely, but not yet rigorously proven, that this first photooxidation site remains a Mn ligation site for the intact Mn cluster. Mutagenesis studies have implicated the D1 protein residue aspartate-170 in high affinity binding of this photooxidizable Mn(II). 4 There have been relatively few EPR investigatio...

The redox-active tyrosines, Y(Z) and Y(D), of Photosystem II are oxidized by P680+ to the neutral tyrosyl radical. This oxidation thus involves the transfer of the phenolic proton as well as an electron. It has recently been proposed that tyrosine Y(Z) might replace the lost proton by abstraction of a hydrogen atom or a proton from a water molecule bound to the manganese cluster, thereby increasing the driving force for water oxidation. To compare and contrast with the intact system, we examine here, in a simplified Mn-depleted PSII core complex, isolated from a site-directed mutant of Synechocystis PCC 6803 lacking Y(D), the role of proton transfer in the oxidation and reduction of Y(Z). We show how the oxidation and reduction rates for Y(Z), the deuterium isotope effect on these rates, and the Y(Z)* - Y(Z) difference spectra all depend on pH (from 5.5 to 9.5). This simplified system allows examination of electron-transfer processes over a broader range of pH than is possible with the intact system and with more tractable rates. The kinetic isotope effect for the oxidation of P680+ by Y(Z) is maximal at pH 7.0 (3.64). It decreases to lower pH as charge recombination, which shows no deuterium isotope, starts to become competitive with Y(Z) oxidation. To higher pH, Y(Z) becomes increasingly deprotonated to form the tyrosinate, the oxidation of which at pH 9.5 becomes extremely rapid (1260 ms(-1)) and no longer limited by proton transfer. These observations point to a mechanism for the oxidation of Y(Z) in which the tyrosinate is the species from which the electron occurs even at lower pH. The kinetics of oxidation of Y(Z) show elements of rate limitation by both proton and electron transfer, with the former dominating at low pH and the latter at high pH. The proton-transfer limitation of Y(Z) oxidation at low pH is best explained by a gated mechanism in which Y(Z) and the acceptor of the phenolic proton need to form an electron/proton-transfer competent complex in competition with other hydrogen-bonding interactions that each have with neighboring residues. In contrast, the reduction of Y(Z)* appears not to be limited by proton transfer between pH 5.5 and 9.5. We also compare, in Mn-depleted Synechocystis PSII core complexes, Y(Z) and Y(D) with respect to solvent accessibility by detection of the deuterium isotope effect for Y(Z) oxidation and by 2H ESEEM measurement of hydrogen-bond exchange. Upon incubation of H2O-prepared PSII core complexes in D2O, the phenolic proton of Y(Z) is exchanged for a deuterium in less than 2 min as opposed to a t(1/2) of about 9 h for Y(D). In addition, we show that Y(D)* is coordinated by two hydrogen bonds. Y(Z)* shows more disordered hydrogen bonding, reflecting inhomogeneity at the site. With 2H ESEEM modulation comparable to that of Y(D)*, Y(Z)* would appear to be coordinated by two hydrogen bonds in a significant fraction of the centers.

An ESEEM (electron spin−echo envelope modulation) spectroscopic study employing a series of 2H-labeled alcohols provides direct evidence that small alcohols (methanol and ethanol) ligate to the Mn cluster of the oxygen evolving complex (OEC) of Photosystem II in the S2-state of the Kok cycle. A numerical method for calculating the through-space hyperfine interactions for exchange-coupled tetranuclear Mn clusters is described. This method is used to calculate hyperfine interaction tensors for protons [deuterons] in the vicinity of two different arrangements of Mn ions in a tetranuclear cluster:  a symmetric cubane model and the EXAFS-based Berkeley “dimer-of-dimers” model. The Mn−H distances derived from the spectroscopically observed coupling constants for methanol and ethanol protons [deuterons] and interpreted with these cluster models are consistent with the direct ligation of these small alcohols to the OEC Mn cluster. Specifically, for methanol we can simulate the three-pulse ESEEM time domain pattern with three dipolar hyperfine interactions of 2.92, 1.33, and 1.15 MHz, corresponding to a range of maximal Mn−H distances in the models of 3.7−5.6 Å (dimer-of-dimers) and 3.6−4.9 Å (symmetric cubane). We also find evidence for limited access of n-propanol, but no evidence for 2-propanol or DMSO access. Implications for substrate accessibility to the OEC are discussed.

Photosystem II (PS II) carries out the photochemical extraction of electrons from water, forming molecular oxygen in the process. The catalytic center for this essential four-electron oxidation reaction is known as the oxygen-evolving complex (OEC). A broad radical electron paramagnetic resonance (EPR) signal can be photogenerated in photosystem II following a variety of treatments that inhibit oxygen evolution. [1][2][3][4][5] The radical has line widths between 90 and 240 G (full width at half-maximum) depending on the specific treatment, and has a characteristic "split" line shape ( Figure 1A). The molecular origin of this signal has been intensely debated, 6,7 with possibilities including oxidized histidine, 8-10 partially oxidized substrate water, 11 or the redox-active tyrosine, Y Z , that is the sole electron transfer intermediate between the photooxidized P 680 + chlorophyll moiety and the tetranuclear Mn cluster at the heart of the OEC. 4,12,13 The species giving rise to the split EPR signal is in close proximity (approximately 4.5 Å 12 ) to the Mn cluster, which accounts for its broad line width. 8,12 We report the results of continuous wave and pulsed EPR experiments performed on this signal in photosystem II particles in which tyrosine is specifically labeled with deuterons in the nonexchangeable hydrogen positions. These experiments conclusively demonstrate that the split EPR signal originates from tyrosine and provide strong support for new metalloradical mechanistic models for oxygen evolution invoking tyrosine Y Z in proton 7,12,14 or hydrogen atom 15,16 abstraction from substrate water bound to the Mn cluster of the OEC.Cells of Synechocystis PCC 6803 were grown photoautotrophically on either deuterated tyrosine (d 7 -Tyr) or natural abundance tyrosine (h 7 -Tyr). 17 Oxygen-evolving PS II core complexes 18 were washed with pH 5.5 acetate buffer 5 (40 mM Mes-NaOH, 500 mM acetate, and 300 mM sucrose) and then centrifuged (266000g, 1 h) in the presence of 8% polyethyleneglycol (final concentration). 19 The complexes were resuspended in the acetate buffer, the chlorophyll concentration was adjusted to 2-3 mg/mL, and 0.3 mM potassium ferricyanide (final concentration) was added. The acetate-treated PS II samples were frozen during illumination 20 in order to generate the split-signal radical. The radical was later eliminated for baseline subtraction purposes by dark annealing the sample at 0°C for 30 min. CW-EPR spectra were recorded on a Bruker ER200D spectrometer. Electron-spin echo envelope modulation (ESEEM) experiments were performed with a 1 kW pulsed EPR spectrometer. 21 Three-pulse ESEEM experiments were performed by incrementing the time T in the stimulated echo sequence: π/2-τ-π/2-T-π/2-τ-stimulated echo. 22 Previously, a split EPR signal in Synechocystis PS II particles has been reported following Ca 2+ -depletion. 24 However, the 40 G wide signal from tyrosine Y D • (as measured under pulsed EPR field swept conditions) overlaps significantly with this weak 90 G wide signal, making it ...

Photosystem II (PS 11) contains two symmetry-related redoxactive tyrosine residues designated YD (D2-Tyr160) and YZ .' The conventional view of the function of tyrosine YZ is as an electron transfer intermediate between the tetranuclear Mn cluster where water is oxidized and the photooxidized chlorophyll moiety P&o.2 Our recent ESE-ENDOR study indicates a 4.5 A separation between the Mn cluster and

The catalytic cycle of numerous enzymes involves the coupling between proton transfer and electron transfer. Yet, the understanding of this coordinated transfer in biological systems remains limited, likely because its characterization relies on the controlled but experimentally challenging modifications of the free energy changes associated with either the electron or proton transfer. We have performed such a study here in Photosystem II. The driving force for electron transfer from TyrZ to P680•+ has been decreased by ~ 80 meV by mutating the axial ligand of P680, and that for proton transfer upon oxidation of TyrZ by substituting a 3-fluorotyrosine (3F-TyrZ) for TyrZ. In Mn-depleted Photosystem II, the dependence upon pH of the oxidation rates of TyrZ and 3F-TyrZ were found to be similar. However, in the pH range where the phenolic hydroxyl of TyrZ is involved in a H-bond with a proton acceptor, the activation energy of the oxidation of 3F-TyrZ is decreased by 110 meV, a value which correlates with the in vitro finding of a 90 meV stabilization energy to the phenolate form of 3F-Tyr when compared to Tyr (Seyedsayamdost et al., 2006, JACS 128:1569–79). Thus, when the phenol of YZ acts as a H-bond-donor, its oxidation by P680•+ is controlled by its prior deprotonation. This contrasts with the situation prevailing at lower pH, where the proton acceptor is protonated and therefore unavailable, in which the oxidation-induced proton transfer from the phenolic hydroxyl of TyrZ has been proposed to occur concertedly with the electron transfer to P680•+. This suggests a switch between a concerted proton/electron transfer at pHs < 7.5 to a sequential one at pHs > 7.5 and illustrates the roles of the H-bond and of the likely salt-bridge existing between the phenolate and the nearby proton acceptor in determining the coupling between proton and electron transfer.

We report the high-frequency (139.5 GHz) electron paramagnetic resonance (EPR) spectrum of the tyrosyl radical of photosystem II. A rhombic powder pattern with principal g values g 1 = 2.007 82, g 2 = 2.004 50, and g 3 = 2.002 32 is observed. The well-defined turning points and the value of the largest principal g value are indicative of ordered hydrogen bonding to the tyrosyl phenyl oxygen. Hyperfine structure is resolved on all three turning points. Proton hyperfine couplings obtained from the simulation of the 139.5 GHz EPR spectrum are in good agreement with X-band electron spin echo−electron nuclear double resonance studies. The high-frequency EPR spectrum was acquired under conditions of saturation in which the dispersion signal is detected. Proper replication of the high-frequency EPR spectral features is only achieved in simulations which account for the line shapes characteristic of saturated dispersion signals. Comparison of the spectrum with spectra of non-hydrogen bonded tyrosyl radicals indicates that the largest principal g value (g 1), oriented along the C−O bond, is sensitive to hydrogen bonding at the phenyl oxygen. Density functional calculations indicate that the decreased downfield shift in g 1 from the free electron g value with increasing hydrogen bond strength arises from both a decreased spin density on the phenyl oxygen5−30% over a range of reasonable hydrogen bond distances (2.0−1.1 Å)and an increased splitting between ground state and excited state singly occupied molecular orbitals.

Previously, using acetate deuterated in the methyl hydrogen positions, we showed that acetate binds in close proximity to the Mn cluster/Y(.)(z) tyrosine dual spin complex in acetate-inhibited photosystem II (PSII) preparations exhibiting the "split" EPR signal arising from the S(2)-Y(.)(z) interaction [Force, D. A.; Randall, D. W.; Britt, R. D. Biochemistry 1997, 36, 12062-12070]. By using paramagnetic NO to quench the paramagnetism of Y(.)(z), we are able to observe the ESEEM spectrum of deuterated acetate interacting with only the Mn cluster. A good fit of the ESEEM data indicates two (2)H dipolar hyperfine couplings of 0.097 MHz and one of 0.190 MHz. Modeling of these dipolar interactions, using our "dangler" 3 + 1 model for the S(2)-state of the Mn cluster, reveals distances consistent with direct ligation of acetate to the Mn cluster. As acetate inhibition is competitive with the essential cofactor Cl(-), this suggests that Cl(-) ligates directly to the Mn cluster. The effect of acetate binding on the structure of the Mn cluster is investigated by comparing the Mn-histidine coupling in NO/acetate-treated PSII and untreated PSII using ESEEM. We find that the addition of acetate and NO does not affect the histidine ligation to the Mn cluster. We also investigate the ability of acetate to access Y(.)(z) in Mn-depleted PSII, a PSII preparation expected to be more solvent accessible than intact PSII. We detect no coupling between Y(.)(z) and acetate. We have previously shown that small alcohols such as methanol can ligate to the Mn cluster with ease, while larger alcohols such as 2-propanol, as well as DMSO, are excluded [Force, D. A.; Randall, D. W.; Lorigan, G. A.; Clemens, K. L.; Britt, R. D. J. Am. Chem. Soc. 1998, 120, 13321-13333]. We probe the effect of acetate binding on the ability of methanol and DMSO to bind to the Mn cluster. We find that methanol is able to bind to the Mn cluster in the presence of acetate. We detect no DMSO binding in the presence of acetate. Thus, acetate binding does not increase the affinity or accessibility for DMSO binding at the Mn cluster. We also explore the possibility that the acetate binding site is also a binding site for substrate water. By comparing the ratioed three-pulse ESEEM spectra of a control, untreated PSII sample in 50% D(2)O to an NO/acetate-treated PSII sample in 50% D(2)O, we find that the binding of acetate to the oxygen evolving complex of photosystem II displaces deuterons bound very closely to the Mn cluster.