Structural Effects of Ammonia Binding to the Mn4CaO5 Cluster of

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Structural Effects of Ammonia Binding to the MnCaO Cluster of Photosystem II 4

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David A. Marchiori, Paul H. Oyala, Richard J. Debus, Troy A. Stich, and R. David Britt J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b11101 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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The Journal of Physical Chemistry

Structural Effects of Ammonia Binding to the Mn4CaO5 Cluster of Photosystem II

AUTHORS: David A. Marchiori1, Paul H. Oyala1, Richard J. Debus2, Troy A. Stich1, and R. David Britt1,* AFFILIATION: 1. Department of Chemistry, University of California, Davis, Davis CA 95616 2. Department of Biochemistry, University of California, Riverside, Riverside CA 92521 E-MAIL: [email protected]

Abstract The Mn4CaO5 oxygen-evolving complex (OEC) of photosystem II catalyzes the light-driven oxidation of two substrate waters to molecular oxygen. ELDOR-detected NMR along with computational studies indicated that ammonia, a substrate analogue, binds as a terminal ligand to the Mn4A ion trans to the O5 µ4 oxido bridge. Results from electron spin echo envelope modulation (ESEEM) spectroscopy confirmed this and showed that ammonia hydrogen bonds to the carboxylate sidechain of D1-Asp61. Here we further probe the environment of OEC with an emphasis on the proximity of exchangeable protons, comparing ammonia-bound and unbound forms. Our ESEEM and electron nuclear double resonance (ENDOR) results indicate that ammonia substitutes for the W1 terminal water ligand without significantly altering the electronic structure of the OEC.

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The oxygen-evolving complex (OEC) of photosystem II (PSII), a Mn4CaO5 cluster (Figure 1A), catalyzes the light-driven four-electron oxidation of two substrate waters, yielding four electrons and protons and a single molecular oxygen. The four electrons are transferred stepwise from the OEC through a series of discrete “S-state” intermediates S0-S4.1 O2 is released from the OEC following formation of the most oxidized S4 state, with the four water-derived electrons returning the OEC to its most reduced state, S0.


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Figure 1. (A) X-ray structure of the OEC poised in the S1-state (PDB ID 4UB6).2 Atom colors: purple, Mn; green, Ca; and red, O. (B) Proposed core OEC structures of “open” and “closed” conformations of the S2-state,3 including the location of the unique Mn(III) ion. In the dark-stable S1 state, a heterometallic CaMn3O4 cuboidal cluster4 is connected to a fourth “dangler” manganese (Mn4A) via two oxido bridges (O4 and O5). In this relatively reduced S1 state, with an electronic configuration of two Mn(III) ions and two Mn(IV) ions,5, 6 four water molecules are directly bound to metals within the OEC: two to the calcium ion that forms a vertex of the CaMn3O4 cuboidal cluster, and two to the dangler manganese Mn4A.2 Xray diffraction and spectroscopy and associated QM/MM calculations indicate Mn4A and Mn1D as the likely Mn(III) sites in the S1 state.2, 7, 8 The subsequent single-electron photooxidation of the OEC generates the S2 state, with three Mn(IV) ions and a unique Mn(III).9,10 In the laboratory this photooxidation is often implemented by a low-temperature (185–200 K) illumination procedure.11 Following such a lowtemperature illumination, the S2 state exists in one of two electron-spin conformers: an S = 1/2 “open” form or an S = 5/2 “closed” form (Figure 1B), which exhibit X-Band (9 GHz) CW EPR signals at g = 2 (typically referred to as the multiline signal (MLS)) and g = 4.1, respectively.12, 13 The conversion from the open to the closed form of the S2 state is believed to involve breaking of the bond between Mn4A and O5, allowing O5 to become a vertex of the cuboidal part of the OEC and a shifting of the unique Mn(III) from Mn1D14 in the g = 2 multiline signal form to the Mn4A dangler position in the g = 4.1 form.3 As-isolated PSII from spinach present a significant amount of the S = 5/2 g = 4.1 signal in the S2 state, yet little if any is present in as-prepared samples of PSII from Synechocystis (cf. CW EPR spectra presented in Figure S1).15 Thus, in addition to chemical means (e.g., treatment with fluoride16 or ethanol12) to modulate the relative populations of the open and closed 3 ACS Paragon Plus Environment


conformations, there are differences between PSII from different organisms that can affect this equilibrium. For example, a recent computational study examining the interaction of methanol with the OEC found that a single amino acid substitution, D1-Asn87 in cyanobacteria versus D1Ala87 in spinach, may be responsible for the difference in the effect methanol has on the magnetic properties of PSII from cyanobacteria and higher plants.17 Treatment of PSII with various small water analogs such as methanol and ammonia has been helpful in determining which of these four water ligands, or others that may bind to the OEC at later S-states, are the actual two substrate waters that are oxidized to form O2.18-23 Both 16

O and 18O are nonmagnetic nuclei, but water binding can be examined via EPR by measuring

hyperfine couplings to the magnetic 17O nucleus introduced in 17O-labeled water,24-26 although this I = 5/2 nucleus has the disadvantages of low gyromagnetic ratio, large quadrupolar broadening, and high cost. The I = 1 deuteron (2H or “D”) can be introduced via D2O, though this magnetic nucleus will be scrambled over any exchangeable hydrogen sites accessible to the added D2O. Pulse EPR experiments comparing normal H2O buffer PSII samples to D2O exchanged PSII have been performed,23, 27, 28 including in this work, in order to determine what exchangeable and non-exchangeable hydrogen sites are in the immediate vicinity of the OEC. Ammonia binding has been extensively studied with a number of EPR methods.20, 21, 29, 30 The I = 1/2 15N nucleus can be substituted for the naturally abundant I = 1 14N nucleus, both of which provide good and distinguishable pulse EPR targets. Ammonia inhibits the OEC competitively with chloride, likely at chloride sites determined in the X-ray structure. 31-33 This chloride-dependent ammonia site does not affect the electronic structure of the OEC as low temperature illumination of such ammonia-treated samples yields EPR spectra that are indistinguishable from those for untreated PSII. Annealing the sample at a higher temperature


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(250 K) alters the 55Mn hyperfine splittings, which serves as one spectroscopic indication of a more intimate interaction by ammonia with the OEC.29 This chloride-independent ammonia binding site, which gives rise to the altered MLS, is non-competitive with substrate water, yet can slow steady-state oxygen evolution.34, 35 Early pulse EPR electron spin echo envelope modulation (ESEEM) spectroscopic results showed that ammonia was directly bound to manganese of the OEC by virtue of a significant isotropic hyperfine (Aiso = 2.29 MHz for 14N, 3.22 MHz for 15N) interaction (HFI) to the OEC in the S2-state MLS form.30 On the basis of the ESEEM-measured high asymmetry of the quadrupole interaction (NQI) of the I = 1 14N ammonia nucleus, it was suggested that ammonia could be bound as deprotonated bridging amido between two Mn ions or between a Mn ion and Ca ion, although it was noted that this asymmetry could also result from strong hydrogen bonding.30 A recent kinetics study by Vinyard and Brudvig suggested that the measured decrease in the reduction potential of the OEC upon ammonia binding is too large to support ammonia binding as a terminal ligand to a Mn(IV).36 Instead, they favor ammonia as a bridging ligand in place of O5, ruling out the possibility of O5 as a substrate water in a radical coupling scheme proposed by Siegbahn.37 However, work by Navarro et al.21 demonstrated that the 17O HFI for solvent exchangeable oxygen sites are only modestly perturbed by ammonia addition. Substrate exchange kinetics monitored by 16O2/16O18O/18O2 time-resolved membrane inlet mass spectrometry similarly were weakly perturbed by ammonia addition, suggesting ammonia binding in place of W1 to Mn4A. Subsequent ESEEM studies tested this specific W1 binding proposal.20 Guided by the crystal structure of untreated PSII, D1-D61− a hydrogen-bonding partner of W1− was mutated to the non-hydrogen-bonding residue, alanine (D1-D61A). 14N ESEEM of ammonia treated 5 ACS Paragon Plus Environment


samples revealed the ammonia hyperfine couplings are nearly identical in wild type and D1D61A. Further, the nuclear quadrupole parameters (e2qQ) also closely match between wild type and D1-D61A. However, the nuclear quadrupole asymmetry parameter (η) is dramatically altered upon mutation. In wild type, this asymmetry parameter is relatively rhombic (η = 0.42), matching the calculated D1-D61 hydrogen bonding interaction,38, 39 yet in the D1-D61A mutant this asymmetry parameter is axial (η = 0.04) characteristic of a terminal ammonia ligand of C3v symmetry. This dramatic collapse of the asymmetry parameter upon mutation of the D1-D61 to alanine demonstrates the hydrogen bonding interaction between ammonia and D1-D61. This strongly suggests ammonia binds to the dangler Mn4A ion in place of W1 (Figure 2A).

Figure 2. Comparison of two different models of the ammonia-bound S2 state. (A) BS-DFT model of the ammonia-bound S2 state by Lohmiller et al. with direct displacement of W1 by NH3.39 (B) QM/MM model of the ammonia-bound S2 state by Askerka et al. via a “carousel” displacement of W1 and W2.40

Recently reported room-temperature crystal structures (2.25 Å and 2.8 Å resolution) acquired using femtosecond X-ray pulses from a free-electron laser (XFEL) showed changes in 6 ACS Paragon Plus Environment

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the electron density at W2 for ammonia-treated PSII subjected to two flashes of laser light (i.e., enriched in S3) compared to that of native PSII.41 Based on these electron density changes the authors conclude that ammonia is binding in place of W2 in the S3 state despite the fact that one cannot directly discriminate between a water oxygen and an ammonia nitrogen at these resolutions. Additionally, the authors do not observe structural changes to the OEC or surrounding complement of electron acceptors seen in a more recent, 2.35 Å XFEL structure of PSII poised in the S3 state.42 At present, there is not enough evidence to unambiguously assign the binding site of ammonia in the S3 state. A separate ammonia binding model has also been introduced by the Brudvig and Batista groups.40 This QM/MM-produced model, aimed to replicate Mn···Mn distances of ammoniatreated PSII determined by EXAFS spectroscopy43, concluded that NH3 binds in addition to W1 and W2 (Figure 2B). In this model, W1 and W2 move toward the cuboidal part of the cluster in a “carousel” fashion, the bond between O5 and Mn4A is broken, and the OEC adopts a structure akin to the closed conformation (Figure 1B, right). This carousel mechanism of ammonia binding has been proposed as an analogous model for the S2 to S3 transition and overall O—O formation mechanism44, 45 and thus testing its veracity is essential. Here we highlight some of the key structural differences between the W1 “direct displacement” model (Figure 2A) and carousel model (Figure 2B). The geometric structure of the W1 direct displacement model largely preserves the Mn···Mn distances and Mn—O—Mn bond angles proposed for the open structure of PSII in the low-spin S2 state (cf. Figure 2A and Figure 1B). The carousel model of Askerka et al. is similar to the S = 5/2 closed structure (cf. Figure 2B and Figure 1B), in which the Mn4A bond to O5 is broken.3, 40 The carousel model retains W1 upon the addition of ammonia to Mn4A. Thus there are three additional protons when 7 ACS Paragon Plus Environment


compared to untreated PSII in the S2 state, one of which resides on O5. The direct displacement model adds a single proton when compared to untreated PSII in the S2 state. In this present report, we employ 1H ENDOR and 2H ESEEM to examine the solvent environment of the OEC in the absence and presence of ammonia. We use these results as well as 55Mn ENDOR to evaluate the different ammonia-binding models introduced above. We find that upon the addition of ammonia, the solvent environment of the OEC is unperturbed. Additionally, the 55Mn hyperfine tensors are only modestly perturbed by the introduction of ammonia, suggesting the geometric structure of the OEC is not affected by ammonia binding. Collectively, these data favor a simple W1 displacement upon ammonia binding to the OEC. Methods. Construction of D1-D61A and WT* Synechocystis Mutants and Propagation of Cultures. Construction of the D1-D61A mutation has been described previously: the mutation was introduced into the psbA-2 gene of Synechocystis sp. PCC 6803 and transformed into a host strain of Synechocystis that lacks all three psbA genes and contains a hexahistidine-tag (His-tag) fused to the C-terminus of CP47.46, 47 Single colonies were selected for their ability to grow on solid media containing 5 µg/mL kanamycin monosulfate. Solid media contained 5 mM glucose and 10 µM DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). The DCMU and antibiotic were omitted from the liquid cultures. The wild-type* (WT*) control strain was constructed in the same manner as the D1-D61A mutant, but with a transforming plasmid with no mutation. The designation “wild-type*” differentiates this strain from the native wild-type strain that contains all three psbA genes and is sensitive to antibiotics. Large-scale liquid cultures (each consisting of three 7-L cultures held in glass carboys) were propagated as described previously.48 To verify the integrity of the mutant cultures that were harvested for the purification of PSII core complexes, 8 ACS Paragon Plus Environment

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an aliquot of each culture was set aside, and the sequence of the relevant portion of the psbA-2 genes was obtained after PCR amplification of genomic DNA.46 No traces of the wild-type codon were detected in any of the mutant cultures. Purification of Synechocystis PSII Core Complexes. Oxygen-evolving PSII core complexes were purified as described previously in buffer A [1.2 M betaine, 10% (v/v) glycerol, 50 mM MESNaOH (pH 6.0), 20 mM CaCl2, 5 mM MgCl2, 1 mM EDTA, and 0.03% (w/v) n-dodecyl β-Dmaltoside] containing 50 mM histidine.49 The purified PSII core complexes were concentrated to approximately 1 mg of Chl per mL, as described previously, and frozen as 0.5 mL aliquots in liquid N2.49 Preparation of Synechocystis EPR Samples. Each sample (0.5 mL) was concentrated in a Centricon-100 centrifugal concentrator (Millipore Corporation, Bedford, MA) to 60 µL. The concentrated sample was diluted with 1 mL of a solution containing [1.2 M betaine, 10% (v/v) glycerol, 20 mM CaCl2, 5 mM MgCl2, 1 mM EDTA, and 0.03% (w/v) n-dodecyl β-D-maltoside], and then reconcentrated to 60 µL. The reconcentrated sample was then diluted with 1 mL of buffer A containing 50 mM HEPES-NaOH (pH 7.5) and 1 mM PPQB (added from a stock of 100 mM PPQB in DMSO). For NH4Cl-treated samples, this pH 7.5 buffer also contained either 14

NH4Cl or 15NH4Cl (99% 15N enrichment, Cambridge Isotope Laboratories, Andover, MA) with

a resulting free-base NH3 concentration of 2 mM. The sample was again concentrated to 60 µL, transferred to a quartz EPR tube (3.8 mm O.D. for X-band EPR experiments, and 2.4 mm O.D. for Q-band experiments) under dim green light, dark-adapted for 30–45 min in total darkness on ice, and frozen in liquid N2. Spinach PSII sample preparation. PSII-enriched membranes from market-fresh spinach were purified according to the method of Berthold, Babcock, and Yocum, modified to remove



adventitiously bound Mn(II) using 5 mM CaCl2 and 1 mM EDTA.50-52 Membranes were centrifuged for 20 minutes at 30,000×g, and the pellets were resuspended in SHNCE buffer (400 mM sucrose, 40 mM HEPES-NaOH (pH 7.5), 10 mM NaCl, 5mM CaCl2, 1 mM EDTA) and repelleted by further centrifugation. This resuspension and centrifugation was repeated once. The washed pellets were then resuspended in a final buffer containing either 100 mM natural abundance NH4Cl, 15NH4Cl (99% 15N enrichment, Cambridge Isotope Laboratories, Andover, MA), or no added NH4Cl. The artificial electron acceptor phenyl-p-benzoquinone (PPBQ) was added from a 250 mM stock in DMSO to a final concentration of 1 mM, and ethanol was added to 5% (v/v) final concentration to favor the S = 1/2 S2 state for 55Mn ENDOR experiments.18 Membranes were centrifuged a final time and the pellets were loaded into quartz EPR tubes. Deuteration of solvent-exchangeable sites within PSII was accomplished by centrifuging the membranes at 30,000×g for 20 minutes and resuspending the resultant pellets in SHNCE buffer (pH = 7.5; pD = 7.1 prepared in deuterium oxide 99.9% 2H enrichment, Cambridge Isotope Laboratories, Andover, MA). Upon resuspension, membranes were incubated in the dark at 5 °C for 1 hour and repelleted by further centrifugation. The washed pellets were resuspended in a final buffer containing 100 mM natural abundance NH4Cl, 15NH4Cl (99% 15N enrichment, Cambridge Isotope Laboratories, Andover, MA), or no added NH4Cl. Artificial electron acceptor phenyl-p-benzoquinone (PPBQ) was added from a 250 mM stock in DMSO to a final concentration of 1 mM. Membranes were centrifuged a final time and pellets were loaded into into quartz EPR tubes.

Advancement of PSII samples to the S2 state. To trap the S2-state multiline signal prior to binding of ammonia, samples were illuminated for 4 min at 185 K using a liquid nitrogen-cooled gas-


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flow apparatus and a Sylvania ELH 300 W halogen-tungsten lamp (color temperature = 3350 K). This low-temperature illumination has been shown previously in both spinach PSII membrane preparations and PSII cores from Thermosynechococcus elongatus (T. elongatus) to generate a “pre-bound” S2-state which presents an EPR signal with no evidence of alteration by ammonia binding.21, 30, 35 These samples were then annealed for 5 min at 250 K using a previously described procedure.20 EPR Spectroscopy. All continuous wave (CW) EPR spectra were collected at a temperature of 7 K under slow-passage and non-saturating conditions using a Bruker ELEXSYS E500 X-band spectrometer equipped with a super-high Q (SHQE) resonator, an Oxford Instruments ESR900 cryostat, and an ITC-503 temperature controller. All pulse EPR and ENDOR spectroscopic studies were performed at a temperature of 4.5 K using a Bruker ELEXSYS E580 pulse EPR spectrometer equipped with an Oxford-CF935 liquid helium cryostat and an ITC-503 temperature controller. X-band three-pulse ESEEM spectroscopy was performed with a Bruker MS-5 resonator using the pulse sequence: π/2 – τ – π2 – T– π/2 − echo, with T the incremented time. Magnetic field-swept echo-detected EPR spectra were acquired using the pulse sequence π/2 – τ – π − echo. Q-band Davies 55Mn ENDOR was performed with the E580 EPR spectrometer equipped with a 250W RF amplifier (Amplifier Research) and an R.A. Isaacsondesigned cylindrical TE011 resonator adapted for pulse EPR use in an Oxford Instruments CF935 cryostat, and with the pulse sequence π – tRF – πRF – tRF – π/2 – τ – π − echo, where πRF is the optimized RF pulse length and tRF is a fixed delay separating MW and RF pulses.53 Q-band 15N Mims ENDOR was performed with the pulse sequence π/2 – τ – π/2 – tRF – πRF – tRF – π/2 – echo using the same general setup, augmented with a 1 kW ENI A1000 RF amplifier. Specific parameters for field positions, microwave frequencies, pulse, and delay lengths are given in the



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caption of each figure. For pulse EPR experiments, all spectra were acquired at a field position corresponding to g = 1.98, near the maximum of the S2-state signal, yet not overlapping with the EPR spectrum of the persistent tyrosine radical YD•. All spectral simulations were performed in the MATLAB 8.1.0 (R2013a) software package (The Mathworks Inc., Natick, MA) using the EasySpin 5.0.0 toolbox.54, 55 Spin Hamiltonian Formalism. In the S2 state the OEC is composed of Mn(IV) (S = 3/2) and high-spin Mn(III) (S = 2) ions that are exchange-coupled to produce a net ground state S = 1/2 spin system that gives rise to the g = 2 MLS.56, 57 In an uncoupled representation we can write a ) with the individual ion parameters and the six J couplings connecting the four Hamiltonian ( Mn ions: = ∑ ∑

+ , + + + − ∑

(1)

where the terms are, in order: the electron and nuclear Zeeman interactions of the electron spin of site i and nuclear spin center j with the applied magnetic field B0; the hyperfine interaction (HFI) () that couples each electron and nuclear spin; the zero-field splitting (ZFS) tensor (); the nuclear quadrupole tensor (P); and the Heisenberg-Dirac-van Vleck exchange term (), a pairwise exchange coupling term (often approximated as being isotropic) between different paramagnetic centers in the spin system. The coupled electron spin momenta produce a new manifold of coupled spin states S. ). For the coupled S Each of those spin states can be described by a coupled spin Hamiltonian ( = 1/2 spin state without zero field splitting interaction (as for the g =2 state giving rise to the MLS) we can write: =

!"" + ∑

!"",# + +


(2)

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In this representation, !"" is the observed g-matrix and !"", is the observed HFI for each nucleus j in the spin system. These measured g-values and hyperfine parameters are related to the intrinsic magnetic parameters described in equation 1 by projection factors $ for each spin center in the complex. These projection factors, or Clebsch-Gordan coefficients, relate the uncoupled angular momentum tensors of each spin center to the new total electron spin vector and can be calculated following an established methodology.58 The projection factors can be affected by covalency and by the site-specific zero-field splitting tensor Di for each coupled ion if the J/D ratio is small.59 Thus, to determine precisely what the true projection factor is, one needs to measure the metal HFIs of the coupled system and compare them to mononuclear standards.60, 61 Once the projection factors are known, the measured HFI elements !"", can be interpreted in terms of covalency and interspin distance (vide infra). The site-specific hyperfine tensor ( ) can be decomposed into an isotropic component (%&' ) stemming from unpaired electron spin in s-orbitals of the atom and an anisotropic component (T) that is traceless and has both local and non-local contributions. The local anisotropic (Tloc) part results from unpaired spin density in p- or d-orbitals centered on the magnetic nucleus.62 The non-local part is simply a dipolar through-space interaction (Tnon-loc) between the electron and nuclear spins. The total hyperfine interaction is written as [Ax, Ay, Az] = Aiso + [Tx, Ty, Tz]

(3)

In the spin-only point-dipole approximation, where g-matrix anisotropy is ignored and the center of unpaired electron density and the magnetic nucleus are suitably distant from each other (r > 2 Å), Tnon-loc is simply: 3non-loc =

89

:;ℏ

=>? $ @

A B'&C DEF GH


I

(4)


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The distance between the electron and nuclear spin is represented by r, the angle between this

is defined as J and the unpaired spin population on the vector and the applied magnetic field specified spin center is given by $. Values for T can be computed for the nucleus interacting with each manganese spin center using equation 4. This can be converted into a vector of the form [−3, −3, +23] and scaled by the appropriate projection factor. These pairwise dipolar interactions must be rotationally transformed into a common axis system before being added together to give the cluster-wide dipolar hyperfine coupling term for the ligand nucleus. 2

H ESEEM Modulation Depth. The depth of the modulation is governed the number of

hyperfine-coupled nuclei and the facility of nuclear spin level “branching” due to the non-secular contribution to the hyperfine tensor and nuclear quadrupole interactions.63 Specifically, the modulation observed in the time-domain spectrum is given by: V

S (T, LAM (O, 3) ∝ ∑D@∏U LAM J) + ∏U LAM (T, J)I

The term

V3αp( β ) ( q , θ )

(5)

describes the contribution to the modulation by nucleus q with orientation

θ relative to the external static magnetic field. The modulations from all like-oriented nuclei contributing via the same coherence transfer pathway (α or β) are multiplied according to the product rule; then the products from each coherence transfer pathway are added. Finally, this result is summed over all possible orientations to give the total observed echo envelope modulation. In practice, equation 5 essentially provides a “product rule” description of the observed ESEEM modulation of weakly coupled nuclei whereby the modulation patterns for each isolated hyperfine-coupled nucleus can be multiplied together yielding the net modulation pattern.64


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Results and Discussion. The annealed-minus-dark Q-Band 1H Davies ENDOR spectra of PSII from spinach with and without ammonia are displayed in Figure 3 and reveal several weakly coupled protons surrounding the OEC. To simplify the analysis of these spectra, we presume that the 1H hyperfine interactions have axial symmetry—a reasonable assumption for any terminal water ligands to Mn. The perpendicular turning points of each axial ENDOR signal are clearly evident with A⊥ ranging from 1–8 MHz (see Figure S5 which displays the first-derivative or pseudomodulated ENDOR spectrum). The intensity that slopes toward the baseline at the extrema of the ENDOR envelope represents a combination of several A|| turning points.27 These peak positions for untreated PSII are given in Table 1 and compare well to those determined in earlier studies.26-28, 65 Ammonia treatment causes minute changes, on the order of ≈200 kHz, in a number of proton hyperfine couplings. Importantly, the overall width of the 1H ENDOR envelope (≈10 MHz) appears unchanged between the ammonia bound and untreated form precluding the presence of a new, strongly-coupled 1H upon ammonia treatment. These findings are similar to what was observed for PSII from T. elongatus.21 This appears to rule out the presence of an O5 proton, as a proton residing on a µ-oxo bridge is predicted to have A|| turning points >5 MHz26 (Figure S3).



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Figure 3. Top: Q-band 1H Davies ENDOR of the S2 state in the absence (left) and presence (right) of ammonia after annealing at 250 K. Experimental dark-subtracted traces are shown in black and simulation parameters listed in Table S1 are shown in color. Simulation parameters used here are based off of a multipole calculation of DFT produced structures from Lohmiller et al. In the simulation of the S2 state in the presence of ammonia the protons of ammonia are labeled as W1 in the figure caption. Bottom: Q-band 1H Davies ENDOR of the S2 state in the absence (left) and presence (right) of ammonia after annealing at 250 K. Bottom traces represent BBY membranes washed deuterated solvent. Acquisition parameters: microwave frequency = 34.094 GHz; magnetic field = 1230.2 mT; temperature = 4.5 K; π/2 MW pulse length = 64 ns; tau = 400 ns; RF pulse delay = 1000 ns; πRF = 16 µs; rep. time = 5 ms. Table 1. Measured 1H ENDOR Peak Positions in NH3-Treated and Untreated PSII − NH3 (MHz) ± 0.28 ± 0.58 ± 1.08 ± 2.18 ± 3.02 ± 3.83 a

1

+ NH3 ± 0.25 ± 0.52 ± 1.05 ± 2.3 ± 3.3 ± 4.1

H Candidates W3,W4 A⊥ Asp 170 A⊥ W1,W2 A⊥ His332 A⊥ His332 A|| W1,W2 A||

1

Based on measured H HYSCORE values.


Ref. This work 28 26 65 a

This work 27

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We have simulated the 1H ENDOR spectrum for the four OEC-coordinated water molecules seen in the X-ray structure using anisotropic HFI that contain a through-space dipolar contribution which we compute using the coordinates from relevant DFT and QM/MM-predicted structures of PSII (see Supporting Information). For every proton, the dipolar hyperfine interaction with each of the four Mn spin centers is scaled by the spin projection factors reported in Lohmiller et al.,39 transformed into a common reference frame, and summed to yield the net dipolar contribution (Table S1 and colored traces in Figure 3)26 An isotropic HFI of 2 MHz was added to the net dipolar contribution of both W1 and W2 as these waters are bound to the paramagnetic Mn4A ion.27 The summation of these individual contributions—from the two waters bound to Mn4A and the two bound to the calcium ion—provides good agreement with the experimental data set. The most obvious discrepancy is an absence of intensity at ±2 MHz (relative to the 1H Larmor frequency, υ(1H)) that likely results from strongly-coupled, nonexchangeable protons like those bound to the imizadole ring of D1-His332 (see next section).65 Deuterium exchange of PSII samples greatly reduces the intensity of the ENDOR envelope (Figure 3, cf. top and bottom panels). In the presence of ammonia, 1H ENDOR peaks at ±0.28 MHz, ±1.05, and ±4.1 MHz are lost after 1-hour D2O exchange, while those at ±0.52, ±2.3, and ±3.3 MHz remain. Similarly, in the absence of ammonia, the ENDOR peaks at ±0.28, ±1.08, and ±3.83 MHz are lost upon D2O exchange, and peaks at ±0.58, ± 2.18, and ±3.3 MHz remain. The residual peaks in the 1H ENDOR spectrum of D2O-exchanged PSII are almost quantitatively simulated using the multipole calculated hyperfine tensors of the protons on the imidazole ring of D1-His332 and D1-His337, as well as the beta protons of D1-Asp170, D1Asp342, and gamma protons of D1-Glu189 (Figure 3, bottom panels). On the basis of our simulations and previous studies, we assign the ENDOR peaks at ≈ ±1.0 to the A⊥ turning points



of W1 and W2,26 and the features at ±4 MHz to the A|| turning points of W1 and W2,27 while those peaks at ≈ ±0.2 MHz correspond to the A⊥ turning points of W3 and W4. We assign peaks at ≈ ±0.5 and ± 2 MHz, which do not disappear upon exchange in D2O, to the A⊥ turning points of non-exchangeable protons on Asp 17028 and His33265 respectively, while the peaks at ±3 MHz are assigned to the A|| turning points of the non-exchangeable His332 protons. The 1H ENDOR spectra presented above appear to rule out the presence of a new class of strongly hyperfine-coupled proton introduced by the ammonia treatment. However, if ammoniatreatment yields an additional proton with hyperfine coupling similar to another, then ENDOR spectroscopy may not be able to detect it. We use three-pulse ESEEM spectroscopy to overcome this limitation as the modulation depth, i.e. amplitude, of the ESEEM trace can report on the number of equivalent protons contributing to the spectrum (see Methods). The three-pulse ESEEM time domain traces of the annealed-minus-dark difference spectra for both 15N-ammonia treated and untreated membranes in D2O solution are displayed in Figure 4. By virtue of its non-zero NQI and HFI that is near cancellation at X-band,66 the 14N nucleus of ammonia significantly contributes to the spin-echo modulation.21, 30 Therefore, we employed 15NH4Cl to minimize these contributions: the contribution of the 15N nucleus to the spin-echo modulation is a function the non-secular part of the hyperfine tensor, and thus is quite small (see three-pulse ESEEM spectrum of 15N-ammonia bound to PSII in H2O, Figure S2). The time-domain difference spectra feature characteristic deuteron modulations with a period of ≈0.45 µs that damp out after approximately 3 µs. These modulations are absent in spinach membranes prepared in protiated solvent. The 15N-ammonia treated and untreated difference spectra are nearly identical with only a 3% increase in the modulation depth of the first beat upon ammonia binding. According to equation 5, we expect an increase in the number of hyperfine-


Page 18 of 39

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coupled deuterons to increase the modulation depth; therefore, this small change would seem to suggest that the degree of deuteration in ammonia-treated PSII is not much different from untreated PSII. Unlike the ENDOR spectra in Figure 3, the 2H ESEEM spectra only contain contributions from solvent-exchangeable protons. Simulations of the time domain difference spectra (Figure 4, top-right and bottom) were made utilizing the same set of DFT and QM/MM models that produced ENDOR simulations. The 2H HFI was computed simply by scaling those determined for the solvent-exchangeable protons (Table S1, colored blue and Figure S7) by the ratio of the gyromagnetic ratios for 2H and 1H (i.e. γ2H/γ1H ≈ 0.154). Simulations included nuclear quadrupole parameters (η = 0.1 and e2Qq = 0.22 MHz) for each deuterium nucleus based on previously reported values.18, 67

Figure 4. X-band three-pulse ESEEM of the 15N-ammonia-bound S2-state (blue) and untreated S2-state (red) in D2O exchanged BBY membranes prepared from spinach. The top left panel shows the time-domain data after subtraction of a bi-exponential decay function, and the topright and bottom panels display the associated overlaid simulations. The experimental data has 19 ACS Paragon Plus Environment


been scaled to match the modulation amplitude of the first peak of the respective simulation. Simulation parameters are displayed in Table S1. Acquisition parameters: microwave frequency = 9.336 GHz; magnetic field = 336.8 mT; temperature = 4.5 K; π/2 MW pulse length = 12 ns; τ = 208 ns; repetition time = 5 ms.

In these simulations, the added protons required by the carousel model (Figure 2B) induce a pronounced dephasing of the 2H modulation as well as a decrease in the modulation amplitude (Figure 4, bottom-right red trace) caused by the large HFI predicted for the proton bound to O5, whereas the direct displacement model (Figure 2A) has a less pronounced change in modulation depth and phase (Figure 4, bottom-left), in line with our experimental observations. Both DFT and QM/MM models, Lohmiller39 and Askerka68, respectively, provide a quantitatively good fit, i.e. there is a close match in the modulation amplitude for the observed modulations, of the experimental data in the absence of ammonia (Figure S6). In the presence of ammonia, only the DFT model provides a good fit for the experimentally observed modulation amplitudes. Scaling the experimental data to match the modulation amplitude of the first peak for the carousel simulation provides a poor fit of the modulation depth and modulation peak times further suggesting O5 is not protonated upon the addition of ammonia (see also Figure S8, which further considers a range of isotropic HFI for an O5 proton). We have further assessed the effect of ammonia-binding on the electronic structure of the OEC in PSII from Synechocystis and spinach by examining the corresponding changes in the 55

Mn HFI determined by ENDOR spectroscopy at Q-band (Figure 5). In an earlier 55Mn ENDOR

spectroscopic study performed at X-band frequency,60 it was observed that ammonia binding caused a shift of two peaks to lower frequency, indicating a decrease in the average effective 55

Mn HFI—consistent with the decrease in the 55Mn splittings (from 8.7 mT to 6.8 mT) observed

in the X-band CW EPR spectrum.29 Two more recent studies explored the effect of ammonia


Page 20 of 39

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binding on PSII from the thermophilic cyanobacterium T. elongatus using 55Mn ENDOR at Qband. In contrast to PSII from spinach, the 55Mn ENDOR spectrum of low-spin S2 PSII from T. elongatus is virtually unchanged upon ammonia-addition with changes in Aiso of no more than 10 MHz.21, 39

Figure 5. Q-band 55Mn Davies ENDOR of the S2 state in the presence (top traces) and absence of ammonia (bottom traces) after annealing at 250 K. Experimental dark-subtracted traces are shown in black, while simulations using the parameters found in Table 2 are shown in red. Acquisition parameters: D61A-Synechocystis: microwave frequency = 33.915 GHz; magnetic field = 1224.0 mT; temperature = 4.5 K; π/2MW = 40 ns; tau = 380 ns; RF pulse delay tRF = 600 ns; πRF = 7µs; repetition time = 5 ms. WT*-Synechocystis: microwave frequency = 33.920 GHz; magnetic field = 1224.1 mT; temperature = 4.5 K; π/2MW = 40 ns; tau = 380 ns; RF pulse delay tRF = 600 ns; πRF = 7µs; repetition time = 5 ms. Spinach (BBY): microwave frequency = 34.122 GHz; magnetic field = 1228.0 mT; temperature = 4.5 K; π/2MW = 40 ns; tau = 380 ns; RF pulse delay tRF = 600 ns; πRF = 7µs; repetition time = 5 ms.



Page 22 of 39

We have acquired Q-band 55Mn ENDOR spectra of the S2 state for PSII from both higher plants (spinach) and the cyanobacterium Synechocystis as well as the PSII D61A mutant from Synechocystis. The dark (S1)-subtracted ENDOR data with and without NH3 bound are presented in Figure 5. Simultaneous fitting of these ENDOR data and corresponding X-band CW EPR spectra (Figures S1 and S4) yielded the effective 55Mn HFI given in Table 2.

Table 2. Effective 55Mn Hyperfine Tensors (MHz) and g-Tensors for PSII with and without Ammonia. D61A – Synechocystis S2 + NH3 x y ⊥a z(∥) iso aniso D61A – Synechocystis S2 - NH3 x y ⊥a z(∥) iso aniso WT* – Synechocystis S2 + NH3 x y ⊥a z(∥) iso aniso WT* – Synechocystis S2 - NH3 x y ⊥a z(∥) iso aniso Spinach (BBY) S2 + NH3 x y ⊥a z(∥) iso aniso Spinach (BBY) S2 - NH3

g 1.982 1.990 1.986 1.967 1.980 0.019 g 1.983 1.982 1.983 1.972 1.979 0.011 g 1.985 1.960 1.970 1.979 1.975 -0.006 g 1.995 1.972 1.973 1.985 1.984 -0.002 g 1.990 1.964 1.984 1.963 1.972 0.014 g

A1 351 326 339 277 318 62 A1 351 321 336 267 313 69 A1 345 323 334 272 313 62 A1 334 314 334 274 307 50 A1 348 332 336 275 318 65 A1


A2 111 168 140 253 177 -114 A2 138 150 144 290 193 -146 A2 194 189 192 212 198 -21 A2 194 185 192 234 204 -45 A2 194 178 165 208 193 -22 A2

A3 128 77 103 166 124 -64 A3 128 105 117 177 137 -61 A3 150 120 135 190 153 -55 A3 154 169 135 247 190 -86 A3 154 133 201 192 160 -49 A3

A4 199 192 196 241 211 -46 A4 201 184 193 237 207 -45 A4 156 222 189 241 206 -52 A4 222 144 189 261 209 -78 A4 169 214 204 233 205 -42 A4

Page 23 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


x 1.997 328 183 170 230 y 1.972 328 183 170 230 1.983 340 165 199 205 ⊥a 1.985 275 248 243 267 z(∥) iso 1.985 310 205 194 242 aniso 0.000 53 -65 -73 -37 a. Perpendicular hyperfine values are expressed as Y = ((Z + [ )/2). Isotropic hyperfine values are the averages of the indivisual tensor components %&' = (Z + [ + \ )/3. Anisotropic hyperfine values are expressed as ]^%&' = Y - \ .

To evaluate the effect of ammonia binding on the 55Mn HFI, we present the ratios (+NH3:−NH3) that describe changes in the 55Mn HFI for the parallel (A||) and perpendicular (A⊥) components of the each tensor in Table 3. The only rigorous assignment of 55Mn ENDOR features (color coded in Figure 5) to specific ions in the Mn4CaO5 cluster that can be made is for the largest effective 55Mn HFI. This tensor belongs to Mn1D, the lone Mn(III) ion,14 and the corresponding 55Mn ENDOR features are largely unchanged consistent with the reported invariance of the 14N hyperfine of D1-His332, which is a direct reporter of the on-site projection factor for Mn1D.20, 21 The 14/15N HFI of ammonia is a reporter for the projection factor of the Mn4A ion; and this value is essentially unchanged even in the D61A mutant (14N Aiso = 2.36 MHz cf. to WT* in which 14N Aiso = 2.33 MHz (Table S2 and Figure S9)). Therefore, the invariance of the HFI of ligands to Mn4A and Mn1D suggest that the corresponding projection factors are unchanged by the mutation (or that competing changes in the various exchange coupling and on-site zero-field splittings lead to no net change in the local projection factor; however, this seems unlikely). We have no such probes for the projection factors of Mn2C and Mn3B however, even if precise assignments for the 55Mn tensors are incorrect, (e.g., if assignments for Mn2C and Mn3B could be swapped) the following qualitative discussion of the 55

Mn ENDOR results remains valid.



Page 24 of 39

For WT* PSII from Synechocystis and spinach, the changes in the 55Mn HFI induced by ammonia binding appear to be similar (in contrast to the lack of a change observed for PSII from T. elongatus), with a shift in intensity of the two most-weakly hyperfine coupled Mn ions to lower frequencies concurrent with a decrease in the anisotropy of these couplings (Table 2). Little change is observed in the largest Mn coupling assigned to the lone Mn(III) ion (Mn1D) in these systems (see Table 3). That Aiso(14NHis332) is unchanged in all cases studied suggests that the Mn1D projection factor in unaffected by the presence or absence of ammonia. One would expect that if O5 moved nearer to Mn1D, as predicted in the carousel model (Figure 2B), that the zero-field splitting and exchange interactions would be affected; therefore, that the 14N HFI of the D1-His332 is unchanged would seem to favor the direct displacement model.

Table 3. Relative changes in 55Mn effective Y and ∥ Values upon NH3 Binding. Species

Mn1D

Mn2C

Mn3B

Mn4A

Ref

NH3-S2 Y /S2 Y

1.01

0.97

0.88

1.02

This work

NH3-S2 ∥ /S2 ∥

1.04

0.87

0.94

1.02

This work

NH3-S2 Y /S2 Y

1.03

1.01

0.84

1.03

This work

NH3-S2 ∥ /S2 ∥

0.99

0.91

0.77

0.92

This work

NH3-S2 Y /S2 Y

1.04

1.02

0.84

0.83

This work

NH3-S2 ∥ /S2 ∥

1.00

0.84

0.79

0.87

This work

NH3-S2 Y /S2 Y

0.99

1.00

1.01

1.00

39

NH3-S2 ∥ /S2 ∥

1.00

0.91

0.98

1.00

39

D61A - Synechocystis

WT* - Synechocystis

Spinach (BBY)

WT* - T. elongatus


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In contrast to the untreated samples of WT* and spinach PSII, the PSII sample for D1D61A exhibits altered 55Mn splittings in the CW EPR spectrum, and an altered 55Mn ENDOR spectrum in the absence of ammonia. Upon binding of ammonia, however, similar changes to the individual 55Mn HFI tensors are observed, though the magnitude of these changes appears to be somewhat smaller in comparison to WT*. As an aside, the D61A mutant appears to show features from free Mn(II) that grow in only after illumination. The breadth of these Mn(II) signals are much narrower than any of the features resolved in the other PSII variants, with an approximate hyperfine coupling of 253 MHz, typical of free Mn(II), and are attributed to degradation of the cluster in some of the PSII during the cryogenic illumination. Larger changes to the 55Mn hyperfine tensors tentatively assigned to the Mn3B and Mn2C ions are seen upon binding of ammonia to PSII from spinach and Synechocystis than were observed for PSII isolated from T. elongatus. The changes in the 55Mn HFI are similar to those observed when the Ca2+ ion is replaced with Sr2+ in PSII from T. elongatus.39, 61 Comparison of the X-ray crystal structures of native OEC69 and strontium-substituted OEC70 show changes of less than 0.2 Å any of the Mn···Mn distances and almost no change in overall cluster geometry. The 55Mn hyperfine anisotropy of six-coordinate mononuclear Mn(IV)-containing complexes is typically quite small owing to the half-filled nature of the t2g orbital set. However, the magnitude of the observed 55Mn HFI anisotropy for Mn(IV) ions in exchange-coupled Mn(III,IV) dimeric systems is related to the ratio of the zero-field splitting constant (D) of the Mn(III) ion and the exchange coupling (J) between the two spin centers.59 When J is small relative to D, this ratio becomes large and some of the intrinsic 55Mn HFI anisotropy of the Mn(III) ion is manifest in the effective 55Mn HFI tensor of the Mn(IV) ion. The 14N His332 HFI measured for PSII establishes that the Mn1D zero-field splitting is unchanged in all samples.



There is an additional effect, in the case of the tetramanganese OEC, that contributes to Mn(IV) hyperfine anisotropy: the first excited spin state of the cluster (S = 3/2, in this case) is close in energy to the S = 1/2 spin-coupled ground state. As the energy separation between these states (∆) diminishes, the hyperfine anisotropy of all manganese ions can change dramatically (see in particular Figure 3 of reference22). The value of ∆ is most affected by changes in the exchange coupling interactions between the dangling Mn4A and the rest of the cluster. As these three exchange interactions diminish, ∆ is reduced leading to increases in the magnitude of 55Mn hyperfine anisotropy for each ion. Because our observed changes in hyperfine anisotropy are relatively small (Table 2), the changes in ∆ induced by ammonia binding must also be small. Measurements by Lorigan et al.71 indeed show small changes in ∆ upon ammonia treatment (39.7 cm-1 and 33.8 cm-1 for ammonia treated and untreated spinach, respectively). This deduction would seem to exclude the carousel model in which an Mn4A-to-Mn3B exchange pathway is lost. Conclusion. We have previously shown that ammonia binds at the dangling Mn4A as a terminal ligand, forming a hydrogen bond with the carboxylate sidechain of D61.20 However, the complement of water ligands in this ammonia bound form has come into question. In a mechanism proposed by Askerka et al., ammonia binds to the closed (S = 5/2) form of the OEC in the S2 state, which then undergoes redox isomerization to yield the low-spin form; Mn4A maintains both W1 and W2 ligands, though W2 is deprotonated by O5, and the Mn4D—O5 bond is broken.40 Recent DFT calculations suggest that this redox isomerization mechanism is thermodynamically too expensive (barrier = 15−22 kcal/mol).72 Additionally, the modest changes observed via 55Mn ENDOR (Figure 5) in the 55Mn hyperfine tensors (Tables 2 and 3)—


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particularly to those assigned to the Mn1D and Mn4A ions—indicate little change in the overall geometric structure or connectivity between Mn spin centers of the cluster. The breadth of QBand 1H ENDOR spectra (Figure 3) is unaltered by ammonia binding, suggesting that the µ-oxo bridge O5 is not protonated (Figure 2B). Such a proton would be expected to exhibit a large HFI with A|| turning points >5 MHz. Changes in the ENDOR intensity of the more-weakly coupled protons can be modeled with the addition of one more proton of a class similar to that for protons on W1—a finding consistent with the direct displacement model in which the OEC should gain just one more proton. Although ENDOR intensity is not often a reliable metric for analyte concentration, three-pulse ESEEM spectroscopy can be used to count solvent-exchangeable deuterons. The three-pulse ESEEM spectra of PSII are only slightly changed by the ammonia treatment (Figure 4). The three-pulse ESEEM and Q-Band Davies 1H ENDOR suggest that only modest changes occur to the water-binding environment of the Mn4CaO5 cluster upon the addition of ammonia. Together, these data favor the direct displacement of W1 upon ammonia binding to the dangling Mn4 of the OEC. That W1 is displaced by ammonia in the S2 state would preclude W1 as a possible substrate as ammonia is thought to remain bound to the OEC in the S3 and presumably the S4 state.21 With W1 ruled out as a possible substrate we can then conclude that W2, a calcium bound water, and O5 remain possible substrate candidates in the O2 evolution process. A recent XFEL structure of the OEC poised in the S3 state reveals an oxygen bound to Mn1D in close proximity (~1.5 Å) to O5,42 perhaps revealing the OEC just prior to (or during) O—O bond formation. Further study is needed to identify the origins of this oxygen bound to Mn1D, but its presence offers an intriguing target in elucidating the mechanism by which O2 is produced.



Supporting Information. Supporting information includes the following: X-band CW EPR of WT*, D61A, and spinach, 3Pulse ESEEM and simulations, Q-band 1H ENDOR and simulations, Pseudomodulated 1H ENDOR, graphic of waters included in 2H 3-Pulse ESEEM simulations, tabulated Adip 1H HFI values, 14N 3-Pulse ESEEM and associated simulations, and tabulated 14N and NQI parameters

Acknowledgments. This work was funded by the Division of Chemical Sciences, Geosciences, and Biosciences (R.D.B. grant DE-SC0007203 for EPR studies of the OEC; and R.J.D. grant DESC0005291 for mutant construction and sample preparation) of the Office of Basic Energy Sciences of the U.S. Department of Energy.

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Structural Effects of Ammonia Binding to the Mn4CaO5 Cluster of

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