EPR-ENDOR Characterization of (¹⁷O, ¹H, ²H) Water in Manganese Catalase and Its Relevance to the Oxygen-Evolving Complex of Photosystem II

Abstract

The synthesis of efficient water-oxidation catalysts demands insight into the only known, naturally occurring water-oxidation catalyst, the oxygen-evolving complex (OEC) of photosystem II (PSII). Understanding the water oxidation mechanism requires knowledge of where and when substrate water binds to the OEC. Mn catalase in its Mn(III)-Mn(IV) state is a protein model of the OEC’s S₂ state. From ¹⁷O-labeled water exchanged into the di-μ-oxo di-Mn(III,IV) coordination sphere of Mn catalase, CW Q-band ENDOR spectroscopy revealed two distinctly different ¹⁷O signals incorporated in distinctly different time regimes. First, a signal appearing after two hours of ¹⁷O exchange was detected with a 13.0 MHz hyperfine coupling. From similarity in the time scale of isotope incorporation and in the ¹⁷O μ-oxo hyperfine coupling of the di-μ-oxo di-Mn(III,IV) bipyridine model (Usov, O. M.; Grigoryants, V. M.; Tagore, R.; Brudvig, G. W.; Scholes, C. P. J. Am. Chem. Soc. 2007, 129, 11886-11887), this signal was assigned to μ-oxo oxygen. EPR line broadening was obvious from this ¹⁷O μ-oxo species. Earlier exchange proceeded on the minute or faster time scale into a non-μ-oxo position, from which ¹⁷O ENDOR showed a smaller 3.8 MHz hyperfine coupling and possible quadrupole splittings, indicating a terminal water of Mn(III). Exchangeable proton/deuteron hyperfine couplings, consistent with terminal water ligation to Mn(III), also appeared. Q-band CW ENDOR from the S₂ state of the OEC was obtained following multi-hour ¹⁷O exchange, which showed a ¹⁷O hyperfine signal with a 11 MHz hyperfine coupling, tentatively assigned as μ-oxo-¹⁷O by resemblance to the μ-oxo signals from Mn catalase and the di-μ-oxo di-Mn(III,IV) bipyridine model.

Introduction

Efficient solar energy-driven water-oxidation catalysts represent a very promising solution to the problem of generating renewable solar fuels,^{1, 2} cheaply and on a large scale.^3–5 A starting point for such catalytic systems is the photosystem II (PSII) oxygen-evolving complex (OEC).⁶ Composed of four Mn ions and a calcium ion, in addition to a proteinacious ligation environment, the OEC oxidizes in a multistep reaction two water molecules with the energy from four photons of visible light.

Catalytic hypotheses and concomitant model development have been greatly aided by crystallographic structural information on PSII at a resolution down to 1.9 Å.^7–9 However, radiation damage has interfered with identification of the substrate water-binding sites. Identification of these sites is key to understanding the mechanism by which waters are activated for O-O bond formation. EPR spectroscopy can provide information on the binding of water/hydroxo/oxo species to the metal ions in the OEC, and these data may correlate with the substrate waters by comparison to the substrate exchange rates measured by mass spectrometry.^10–14

The paramagnetic S₂ state of the OEC has a characteristic 1.80 T wide, 18+ line CW X-band EPR difference spectrum (S₂ minus S₁) or ‘multiline signal’, centered at g = 2.¹⁵ An early EPR study detected broadening of this signal due to exchange of ¹⁷O from ¹⁷O-labeled water into the OEC.¹⁶ This initial work showed that oxygen from water could interact with the manganese ions in the OEC. More recent studies have provided evidence for ¹⁷O that exchanged from ¹⁷O-labeled water into the OEC. X-band ESEEM (Electron Spin Echo Envelope Modulation) provided evidence for ¹⁷O in the OEC with a hyperfine coupling of ~ 5 MHz.¹⁷ This ESEEM signal was assigned as a terminal water ligated by π bonding to Mn(III),¹⁷ based on the relation of the ¹⁷O hyperfine couplings to those found for terminal water π bonded to low spin ferric cytochrome P450.¹⁸ X-band HYSCORE (Hyperfine Sublevel Correlation) spectroscopy measurements on the OEC exchanged with ¹⁷O-water showed a single pair of structureless features assigned as a ¹⁷O coupling of ≈ 10 MHz.¹⁹ However, while our present submission was in revision, an Addition/Correction was published using a ¹⁵N substitution to show that the 2008 Su et al.¹⁹ nominal ¹⁷O HYSCORE signal actually derived from a ¹⁴N spectral subtraction artifact.²⁰

Di-μ-oxo Mn(III)-Mn(IV) bipyridine had been investigated as a simple chemical model of the OEC which lacks the complexity of that higher nuclearity cluster because it has μ-oxo oxygens but no terminal waters. Mass spectrometry confirmed that the μ-oxo ligands of this Mn(III)-Mn(IV) bipyridine model complex exchanged from ¹⁸O-labeled water.²¹ CW Q-band ENDOR and X-band EPR line broadening experiments revealed a ¹⁷O hyperfine coupling of 12.8 MHz.²² Given the breadth of the ENDOR line shape, we would take this hyperfine coupling as an estimate of the isotropic coupling. Thus, to date, the most reliable ¹⁷O μ-oxo hyperfine coupling has been reported for the di-μ-oxo Mn(III)-Mn(IV) bipyridine model, for which mass spectrometry experiments confirm that the μ-oxo oxygen can exchange with isotopically enriched oxygen from water.²¹

As evidenced by rapid mass spectroscopic detection of ¹⁸O₂ product, two substrate ¹⁸O-waters were found to exchange very quickly into the OEC itself, with product formation times of ~0.01 s and 0.5 s for a fast and a slow site respectively (in the S₂ state).^10–14 It is not clear where these catalytically relevant, rapidly exchanging water binding sites of the OEC are located. There is resonance Raman evidence for a slower oxygen isotope exchange occurring on the order of tens of minutes into a Raman-detectable μ-oxo group localized in the OEC.²³ The mass spectroscopic study on the di-μ-oxo Mn(III)-Mn(IV) bipyridine model which had been dissolved in acetonitrile showed that oxygen ligand exchange into the di-μ-oxo groups from microliter quantities of ¹⁸O-water occurred in about 20 min.²¹ In the same study²¹ terminal water ligand exchange rates of the di-μ-oxo Mn(III)-Mn(IV) terpy model complex could not be resolved, but the μ-oxo ligand exchange time of 400 s from this terpy model compound was the shortest time measured for di-μ-oxo models.

The rate of water exchange into a di-μ-oxo di-Mn system could be affected by an aqueous environment, proteinacious ligation, seclusion from the bulk phase, and electrostatic effects on pKa, all factors not under control in the organic solvent of the model work of Tagore et al.^{21, 24} An improved model, also having the potential for terminal water ligation, could have a similar di-μ-oxo di-Mn center imbedded inside a soluble protein, and Mn catalase (MnCat) was chosen for that model.

The location and environmental character of a di-Mn di-μ-oxo center is known in the proteinaceous system of MnCat; the positions of both bridging oxygens, protein ligands, terminal water, and potentially critical second sphere amino acids are crystallographically known, as shown in Figure 1. MnCat is a 29.6 kDa hexamer forming-protein found in Lactobacillus plantarum²⁵ and also Thermus thermophilus.²⁶ It contains a di-Mn catalytic core with solvent derived aquo, hydroxo²⁷ or μ-oxo bridges,²⁸ depending on the oxidation state of the Mn. Under catalytic conditions, the Mn core oscillates between (II,II) and (III,III) oxidation states to disproportionate peroxide in a ping-pong mechanism.^{29, 30} The enzyme can be oxidized beyond the (III,III) state by treatment with hydrogen peroxide and hydroxylamine^{25, 31} or KIO₄.^{26, 32, 33} This form of the enzyme, while catalytically inactive, is EPR active,^{32, 34–36} producing a 16-line signal at g = 2 which is diagnostic of antiferromagnetically coupled Mn(III)-Mn(IV).^{26, 33} EXAFS of this form of MnCat has also indicated a decrease in the Mn-Mn distance consistent with oxidation of one manganese to Mn(IV),³⁷ and this decrease was consistent with the Mn(III)-Mn(IV) distance found in di-μ-oxo models.³⁸ Advanced paramagnetic resonance studies have already been performed on MnCat in the (III,IV) state,^{37, 39,40} but the ¹⁷O ENDOR signals from potential μ-oxo or terminal water sites of MnCat have not been reported.

Figure 1.

Shown is the local liganding environment of the di-manganese center of MnCat from L. plantarum. The structure is taken from the X-ray crystallographic structure of the Mn(III)-Mn(III) derivative, PDB structure 1JKU.²⁷ Glu178 does not directly ligate the manganese but is within hydrogen bonding distance (2.5 Å) of the liganding water oxygen. The μ labels indicate solvent derived ligands. It is expected that there would be two μ-oxos in the (III,IV) form, and potentially a μ-oxo and μ-OH in the (III,III) state.²⁷

With the goal of learning about the mechanism of water binding and O₂ production in PSII, this work is focused on using ENDOR and EPR spectroscopy to characterize the binding and exchange of ¹⁷O from ¹⁷O-enriched water into both terminal and μ-oxo sites of MnCat. Data for this proteinaceous structural mimic of PSII provides the background to understand water exchange into PSII, its time course, locale, and catalytic relevance.

Materials and Methods

Preparative Methods

MnCat Preparation

MnCat was isolated from L. plantarum according to published procedures.^{25, 41} The superoxidized Mn(III)-Mn(IV) state of MnCat was prepared with minor modifications as previously described.^{25, 27, 31} MnCat was oxidized by redox cycling in the presence of hydrogen peroxide and hydroxylamine. Briefly, MnCat (0.1 mM active sites in 50 mM potassium phosphate buffer pH 7, containing 0.1 mM EDTA) was dialyzed against 1 L of 0.1 mM hydroxylamine and 0.1 mM H₂O₂ at 4 °C for 4 h. The protein was then transferred to 1 L of fresh 0.1 mM H₂O₂ solution (two changes), and finally, dialyzed overnight against 50 mM potassium phosphate buffer, pH 7, containing 0.1 mM EDTA. The sample, concentrated to 90 mg mL⁻¹ (3 mM active sites), was then combined with a known volume of H₂¹⁶O or H₂¹⁷O for CW X-band EPR experiments. For Q-band ENDOR, H₂¹⁷O and glycerol; H₂¹⁶O and glycerol; or D₂O and deuterated glycerol (99% deuterium enrichment, Cambridge Isotope Laboratories, MA) were added. (Glycerol at 50% vol/vol is a glassing agent that prevents aggregation of paramagnetic centers upon freezing.) After addition of reagents, the sample was frozen at 77 K. Exchange time with the added H₂¹⁷O reagent was monitored to differentiate between short exchange periods (1–5 min) and long-term exchange (> 2 hours). H₂¹⁷O was supplied at 90 % enrichment from Cambridge Isotope Laboratories. Exchange of D₂O was accomplished in about a minute and the sample frozen; the sample was then unfrozen, deuterated glycerol added, and refrozen in about 5 minutes.

Photosystem II Preparation

Dark-adapted PSII membranes were prepared by the method of Berthold et al.⁴² The membranes were brought in 2.0 mm i.d., 2.4 mm o.d. quartz EPR-ENDOR tubes to a final concentration of 10–20 mg chl/mL and a ~74 % concentration of exchangeable H₂¹⁷O with a method outlined in Supporting Information, Figure 1S. Following dark adaptation for 24 hours, samples were frozen by plunging in liquid nitrogen. To achieve the S₂ state for both the H₂¹⁷O sample and the H₂¹⁶O control, samples were illuminated for 1 min in an acetone bath at 200 K with a xenon arc lamp.

X-band EPR Methods

Mn-Cat cw X band EPR

Samples were stored at 77 K. CW X-band EPR scans were acquired on a Bruker ELEXSYS E500 EPR spectrometer equipped with a SHQ resonator and an Oxford ESR-900 helium-flow cryostat. The 16-line EPR signal was recorded at 7.5 K with the following instrumental parameters: microwave frequency 9.38 GHz, modulation frequency 100 kHz, modulation amplitude 3 G, microwave power 5 mW.

Photosystem II CW X band EPR

Following creation of the S₂ state by photoillumination, the EPR line widths of S₂ state samples enriched in H₂¹⁷O were compared with those of with non-enriched H₂¹⁶O samples. EPR scans were acquired as for MnCat.

CW Q-band ENDOR Methods

Spectroscopic Methods

CW Q-band (34.1 GHz) ENDOR measurements were performed under dispersion (χ′), rapid passage, field-modulated conditions with a cryogenically tunable TE011 Q-band resonator⁴³ at 2 K as previously reported.^{22, 44, 45} For study of PSII, S₂ samples were transferred from liquid N₂ on a specially designed wand into the He-filled ENDOR cavity in the dark within 3 min of illumination. In this method of CW ENDOR, one monitors the radio-frequency (RF)-induced change in the rapid-passage, 100 kHz field modulated dispersion EPR signal as the frequency of the RF field is swept. RF power is pulsed with 100 μsec on/900 μsec off, with a peak power that is generally 20 W. Through previous experience we have determined that strongly coupled protons (coupling ≥ 4 MHz), ¹⁴N, and ¹⁷O are best resolved with a higher field modulation ≥ 2.5 Gauss p.t.p. (peak-to-peak)^{22, 44, 45} while weakly coupled nuclei, notably deuterons here, are best resolved with smaller field modulation ≤ 0.5 Gauss p.t.p.⁴⁴ The frequencies of ENDOR features were determined from the average frequency of spectra taken with increasing and with decreasing frequency sweeps. CW field-modulated ENDOR has higher sensitivity than pulse methods for broad ENDOR signals, such as those of ¹⁷O.⁴⁶ However, pulse methods, not available to us, appear to give better resolution of small detailed couplings, such as those of deuterons.⁴⁷ We are aware that CW ENDOR, as carried out in pumped liquid helium, can also be subject to baseline artifacts from RF and field modulation-induced heating.

First-Order ENDOR Theory

A nucleus, N, with spin I, e.g., ¹⁷O (I = 5/2) or ¹⁴N (I = 1), can be described by a Spin Hamiltonian of the form:

$$\text{Hnucl}={{}^{\text{N}}\text{A}}_{\text{xx}}{\text{I}}_{\text{x}}{\text{S}}_{\text{x}}+{{}^{\text{N}}\text{A}}_{\text{yy}}{\text{I}}_{\text{y}}{\text{S}}_{\text{y}}+{{}^{\text{N}}\text{A}}_{\text{zz}}{\text{I}}_{\text{z}}{\text{S}}_{\text{z}}+{{}^{\text{N}}\text{P}}_{\text{xx}}{{\text{I}}_{\text{x}}}^2+{{}^{\text{N}}\text{P}}_{\text{yy}}{{\text{I}}_{\text{y}}}^2+{{}^{\text{N}}\text{P}}_{\text{zz}}{{\text{I}}_{\text{z}}}^2+{{}^{\text{N}}\text{g}}_{\text{n}}{\beta }_{\text{n}}\mathbf{H}·\mathbf{I}.$$ (1)

Where β_n is the nuclear Bohr magneton and ^Ng_n is the nuclear g-value (¹g_n = 5.585, ²g_n = 0.8574, ¹⁴g_n = 0.4038, ¹⁷g_n = −0.7572, ¹ν = 52.928 MHz, ²ν = 8.124 MHz, ¹⁴ν = 3.816 MHz, ¹⁷ν = 7.178 MHz at 1.2431 T) for protons, deuterium, ¹⁴N, and ¹⁷O, respectively. ^NA_xx, ^NA_yy, and ^NA_zz are the components of the hyperfine tensor, and for nuclei with I > ½, ^NP_xx, ^NP_yy, and ^NP_zz, are the components of the quadrupolar tensor, where ^NP_xx + ^NP_yy + ^NP_zz = 0. [^NP_zz = (3e²q_zz^NQ/h)/(4I(2I−1)) where e is the electronic charge; h is Planck’s constant; ^NQ is the nuclear quadrupole coupling constant of a particular nucleus; I is the nuclear spin operator, and q_zz is the electric field gradient (EFG) along the “z” direction, a direction which may coincide for ¹⁴N or ¹⁷O with a bonding direction.] This terminology for Spin Hamiltonian parameters follows that used in our recent publications.^{44, 45, 48, 49}

¹⁷O ENDOR Frequencies and Hyperfine Analysis

To first order, the ¹⁷O ENDOR frequencies will be:

$$\begin{array}{l}{{{}^{17}\nu }^{+}}_{\text{ENDOR}}=\mid {}^{17}\text{A}/2+{}^{17}\nu \mid \\ \mid {}^{17}\text{A}/2+{}^{17}\nu \pm 3{}^{17}\text{P}\mid \\ \mid {}^{17}\text{A}/2+{}^{17}\nu \pm 6{}^{17}\text{P}\mid \end{array}$$ (2a)

$$\begin{array}{l}{{{}^{17}\nu }^{-}}_{\text{ENDOR}}=\mid {}^{17}\text{A}/2-{}^{17}\nu \mid .\\ \mid {}^{17}\text{A}/2-{}^{17}\nu \pm 3{}^{17}\text{P}\mid \\ \mid {}^{17}\text{A}/2-{}^{17}\nu \pm 6{}^{17}\text{P}\mid \end{array}$$ (2b)

The ¹⁷ν⁺ _ENDOR branch has been the branch generally observed by Q-band CW rapid passage ENDOR on biologically relevant complexes,^{46, 50–52} including the μ-oxo oxygens of the Mn(III)-Mn(IV) bipyridine dimer.²² For those μ-oxo oxygens of the Mn(III)-Mn(IV) bipyridine dimer, the ¹⁷ν⁻_ENDOR branch is close to zero frequency because of the near cancellation of |¹⁷A/2| ≈ 6.4 MHz and ¹⁷ν = 7.18 MHz at 1.243 T). Relevant details of ¹⁷O quadrupole and anisotropic hyperfine couplings are provided in the discussion and the Supporting Information.

¹⁴N ENDOR Frequencies and Hyperfine Analysis

The first-order expressions for spin 1 ¹⁴N ENDOR frequencies are:

$${{{}^{14}\nu }^{+}}_{\text{ENDOR}}=\mid {}^{14}\text{A}/2\pm 3/2{}^{14}\text{P}+{}^{14}\nu \mid$$ (3a)

$${{{}^{14}\nu }^{-}}_{\text{ENDOR}}=\mid {}^{14}\text{A}/2\pm 3/2{}^{14}\text{P}-{}^{14}\nu \mid$$ (3b)

where ¹⁴A is the hyperfine coupling, ¹⁴P the quadrupolar coupling, and ¹⁴ν is the ¹⁴N nuclear Zeeman frequency. For ¹⁴N nitrogen, as opposed to protons, the hyperfine term, ¹⁴A/2, rather than the nuclear Zeeman term ¹⁴ν, is the dominant term measured by ENDOR. As with ¹⁷O, for CW rapid passage Q-band ENDOR, the ¹⁴ν⁺_ENDOR branch is frequently the branch observed.

Protons/Deuteron Frequencies and Hyperfine Analysis

The frequencies of proton or deuteron ENDOR features, ¹ν_ENDOR or ²ν_ENDOR, center, to first order, at the respective free proton or free deuteron nuclear Zeeman frequency, ¹ν or ²ν, and in the proton or deuteron ENDOR spectra we show the spectra centered at ¹ν or ²ν. For example, taking ¹A as the proton hyperfine coupling, one finds that the frequencies, ¹ν^±_ENDOR, are split away from the nuclear Zeeman frequency by ± 1/2¹A for protons coupled to the electron spin ½ doublet. Proton ENDOR frequencies, occurring as “+” or as “−” Zeeman branches, are:⁵³

$${{{}^1\nu }^{\pm }}_{\text{ENDOR}}=\mid {}^1\nu \pm {}^1\text{A}/2\mid$$ (4)

With the neglect of small quadrupolar terms, a similar expression holds for deuterons:

$${{{}^2\nu }^{\pm }}_{\text{ENDOR}}=\mid {}^2\nu \pm {}^2\text{A}/2\mid$$ (5)

First-order expressions hold here because ¹ν > |¹A/2| and ²ν > |²A/2|.

The strong, largely dipolar coupling to terminal water protons localized on the Mn(III) of the (2-OH-3,5-Cl₂-SALPN)₂ Mn(III)-Mn(IV) complex has previously been resolved by ENDOR-ESEEM measurements.^{40, 54} The theory to explain the terminal water dipolar coupling, called extended dipole theory, incorporates the quantum mechanical projection factors for spin localized on the Mn(III) (S = 2) and Mn(IV) (S = 3/2) where these two ions are an antiferromagnetically coupled S = ½ pair. (The projection factor expresses the spin on an individual metal ion in terms of the total spin of the dimer. See, for example, the discussion and formula A3 on p. 4923, Khangulov et al.⁴⁰) The theory provides the diagonalized hyperfine tensor⁵⁴ where both metal ions contribute in a non-colinear fashion to the dipolar Hamiltonian. A more complete discussion of the extended dipole method applied to ¹⁷O–μ-oxo and to the terminal water protons in MnCat is provided in the Supporting Information.

Results

X-band EPR ¹⁷O Line Broadening of MnCat

The first derivative CW X-band spectra were examined in the ¹⁶O- and ¹⁷O-exchanged states. The extent of broadening from the ¹⁷O ligand is clear in Figure 2, which compares the overlaid line shapes of the ¹⁶O-exchanged and ¹⁷O-exchanged. The broader line width implies ¹⁷O exchanged into the Mn(III)-Mn(IV) coordination sphere.

Figure 2.

CW first derivative X-band EPR spectra are presented for MnCat exchanged in H₂¹⁶O (black) and 90% H₂¹⁷O (red). The inset shows an enlarged view of several hyperfine lines to demonstrate the broadening. The spectra from the MnCat in H₂¹⁶O and 90% H₂¹⁷O have been normalized to the integrated spin count. Samples were incubated for two hours after addition of labeled or non-labeled water before freezing.

CW Q-band ¹⁷O ENDOR of MnCat

CW rapid-passage Q-band ENDOR was obtained for water-derived ligands that exchanged into the active site of MnCat. (Q-band rapid-passage EPR of the 16-line spectra of MnCat are shown in Figure 2S of the Supporting Information.) Figure 3A provides evidence for the appearance after a two hour exchange of ¹⁷O-water of a broad signal labeled ¹⁷O_A, similar in line shape and ENDOR frequency to the ¹⁷O from the μ-oxo oxygens in the di-μ-oxo Mn(III)-Mn(IV) bipyridine model complex.²² The frequency of ¹⁷O_A is 13.7 ± 0.5 MHz, and this ENDOR frequency translates via Eq. 2a into a ¹⁷O hyperfine coupling of 13.0 ± 1.0 MHz. To give insight into the approximate time course and origin for appearance of peaks, the MnCat sample was exchanged with ¹⁷O-water for ~ 1 min, and as shown in Figure 3B, the higher frequency ¹⁷O_A feature near 14 MHz was then not present but features in the 7–11 MHz region were. When ¹⁶O-water was used, a peak in the 6–7 MHz region remained, as shown in Figure 3C. This remaining peak had an ENDOR frequency of 6.5 ± 0.3 MHz, and its ¹⁴N coupling per Eq. 3 for ¹⁴ν⁺ is ¹⁴A = 5.4 ± 0.6 MHz. This coupling is consistent with the coupling of ¹⁴A = 5.75 MHz recently reported by multi-frequency ESEEM and assigned to the histidine nitrogen ligated to Mn(III) of L. plantarum MnCat.³⁹ Although there is evidence from Figure 3C for a broad underlying baseline in the 10–25 MHz region, neither of the peaks, ¹⁷O_A and ¹⁷O_B, was at all evident in this baseline.

Figure 3.

This figure provides a comparison of the ENDOR signals in the 1–30 MHz range from MnCat prepared as follows: A) in 66 %¹⁷O-water with a two hour incubation before freezing; B) in 66% ¹⁷O water with a 1 min incubation before freezing; C) ¹⁶O-water. These spectra were obtained at a magnetic field of 1.243 T. Conditions: microwave power = 0.22 μW, modulation amplitude = 5 G p.t.p., time constant = 20 ms, RF power ≈ 10 W, sweep rate = 6 MHz/s. Spectrum A was the result of 3300 5 s sweeps, and B and C were the result of 1000 5 s sweeps. Spectra A, B, and C were normalized to the number of sweeps and to their respective underlying EPR intensities, and the vertical intensity unit scale is the same for A, B, and C. Inset: The inset was obtained from MnCat exchanged in ¹⁷O-water for 1 minute, but to obtain better resolution of weakly coupled features, a field modulation of 2.5 Gauss p.t.p., a slower sweep of 3 MHz/s over a 1–16 MHz range, and a higher RF power by a factor of 2. The inset spectrum was the result of 1100 5 s sweeps.

Figure 3S in the Supporting Information provides a comparison of spectra from the same sample as that of Figure 3A at two different magnetic fields, H = 1.243 T near the center of the EPR pattern and H = 1.267 T at its high field end. Other than a difference in intensity, the ENDOR spectra at the two fields in Figure 3S are essentially identical. A more highly resolved spectrum for this two-hour ¹⁷O-exchanged sample is shown in the Supporting Information, Figure 4S, where there is still the ¹⁷O_A peak near 14 MHz, but where there is evidence for lower frequency features in the 7–11 MHz region, notably those labeled ¹⁷O_B.

By use of lower field modulation in the CW ENDOR technique, higher resolution of the ¹⁷O_B signal than that in Figure 3B is provided in the inset adjacent to Figure 3B using the sample prepared with 1 min ¹⁷O exchange. The ¹⁷O hyperfine coupling of the ¹⁷O_B peak labeled with an arrow in the inset near 9 MHz was 3.8 ± 0.6 MHz. Above 10 MHz there were two shoulders noted in the inset to 3B with splittings of about 1.3 MHz, while overlap with the ¹⁴N signal prevented additional resolution of any ¹⁷O details below 9 MHz. In the Supporting Information, Figure 5S, a comparison is made of the spectrum of the inset to Figure 3B showing the ¹⁷O_B signals and the MnCat sample prepared with H₂¹⁶O which shows the ¹⁴N signal but not ¹⁷O_B signals.

CW Q-band – Proton and Deuteron ENDOR of MnCat

A comparison of CW proton ENDOR from a sample prepared in protonated buffer to a sample prepared in deuterated buffer provided evidence for exchangeable protons at the active site. Detailed features, especially the starred outlying ones, were best resolved in the first derivative presentation of Figure 4. (The standard absorption-like ENDOR spectra before a derivative was numerically taken are shown as Figure 6S in the Supporting Information.) These outlying features, per Eq. 4, had hyperfine couplings of 17.2 ± 0.4 and 12.2 ± 0.4 MHz, larger than any previously reported from MnCat by frequency-modulated X-band ENDOR of the 1990’s,⁴⁰ but comparable with the largest component of the anisotropic proton hyperfine tensor of terminal water ligated to Mn(III) in the (2-OH-3,5-Cl₂-SALPN)₂ Mn(III)-Mn(IV) dimer.⁵⁴ There may be broad underlying exchangeable features with proton hyperfine couplings (A₃) less than 10 MHz, but such features are obscured by non-exchangeable protons.

Figure 4.

This figure presents the first derivative ¹H-ENDOR spectra, centered at the free proton NMR frequency, ¹ν, to compare exchangeable features from MnCat prepared in protonated solution with MnCat prepared in deuterated solution. Conditions: 1.243 T (¹ν = 52.92 MHz), microwave power = 0.22 μW, field modulation = 5 G p.t.p., time constant = 20 ms, sweep rate = 3 MHz/s, 750 scans for the protonated sample and 250 for the deuterated sample. Inset: ²H-ENDOR of D₂O-exchanged MnCat following subtraction of the underlying spectrum from a protonated MnCat sample. Deuteron ENDOR conditions: (²ν = 8.12 MHz), sweep rate = 1 MHz/s, field modulation = 0.5 G p.t.p., scans = 1000. The inset spectrum is centered at the free deuteron NMR frequency, and the scale above it is the proton frequency scale (obtained with a multiplier of 6.52) corresponding to the deuteron scale. Exchange of D₂O was accomplished in about a minute and the sample frozen; the sample was then unfrozen, deuterated glycerol added, and refrozen in about 5 minutes.

Evidence for exchangeable protons implied that there are corresponding exchangeable deuterons, and so ENDOR from exchangeable deuterons was sought near the deuteron Larmor frequency, ²ν = 8.12 MHz, as shown in the inset to Figure 4. The CW deuteron ENDOR spectrum does not have the resolution or signal to noise of the proton spectrum, but it complements the proton ENDOR information and shows evidence of exchangeable deuterons. Protons with hyperfine couplings less than ~10 MHz are obscured in Figure 4 by non-exchangeable protons; whereas, the associated deuterons with couplings less than about 1.5 MHz (i.e., with a splitting away from ²ν of ~±0.75 MHz) are obvious even at low resolution and occur only from exchanged deuterons.

CW Q-band ¹⁷O and ¹⁴N ENDOR of the S₂ State of PSII

The ¹⁷O information from MnCat provided motivation to obtain corresponding information from the S₂ state of PSII. Twenty four hours of exchange with H₂¹⁷O produced a definite, light-induced ¹⁷O hyperfine interaction from the S₂ state of PSII. A comparison (Figure 5) of S₂ ENDOR signals from PSII samples, respectively exchanged with H₂¹⁶O and H₂¹⁷O, indicated a ¹⁷O ENDOR signal at 12.8 ± 0.5 MHz, similar to that of MnCat here and to that of the previously reported ¹⁷O-bridged Mn^III-Mn^IV bipyridine dimer.²² The ¹⁷O hyperfine coupling, derived via Eq. 2a, was |¹⁷A| = 11 ± 1 MHz. An additional signal occurred at 7.3 ± 0.2 MHz from all S₂ samples, and its hyperfine coupling, assigned to ¹⁴N and interpreted by Eq. 3, is |¹⁴A| = 7.0 ± 0.4 MHz. This coupling is similar to the 7.2 MHz coupling reported from ESEEM studies of the OEC and assigned to the D1-H332 histidine nitrogen ^{55, 56} that is ligated to Mn No. 2 (Numbering system of Sproviero et al.⁵⁷)

Figure 5.

ENDOR spectra of white light-illuminated PSII in H₂¹⁷O (red) and H₂¹⁶O (black) and a dark-adapted sample in H₂¹⁷O (green) at field positions: (A) 1.24 T and (B) & (C) 1.27 T. A light-induced feature (¹⁷ν⁺ENDOR) from the ¹⁷O sample occurs at 12.8 ± 1.0 MHz. Acquisition conditions: field modulation 2.5 G, time constant 82 msec, mw power = 0.28 μW, RF sweep rate 3 MHz/sec, number of scans (A) 1200, (B) 600, and (C) 200. Spectral amplitudes were normalized to the peak labeled 14N. Quoted ENDOR and hyperfine frequencies in text are the average of upward and downward frequency sweeps shown in the Supporting Information, Figure 7S.

Discussion

μ-oxo ¹⁷O Hyperfine Interaction

There are now three structurally related systems into which ¹⁷O has been exchanged from ¹⁷O-water. The first is the di-μ-oxo Mn(III)-Mn(IV) bipyridine complex, known by mass spectroscopic quantitation to exchange both of its μ-oxo oxygens with isotopically enriched ¹⁸O-water.^{21, 24} It has no other oxygen ligands to exchange and, therefore, its ¹⁷O ENDOR signal has to be μ-oxo. Both ENDOR and EPR line broadening of the ¹⁷O-di-μ-oxo Mn(III)-Mn(IV) bipyridine complex showed a ¹⁷O hyperfine coupling of 12.8 MHz.²² For the di-μ-oxo Mn(III)-Mn(IV) bipyridine complex, the time for induction of the ¹⁷O signal by exchange from ¹⁷O-water was of order twenty minutes. The present study on MnCat showed an even longer time for induction of its ¹⁷O signal (¹⁷O_A in Figure 3A), a signal having a very similar hyperfine coupling to that of the di-μ-oxo Mn(III)-Mn(IV) bipyridine complex of 13.0 MHz. MnCat is known to have a di-μ-oxo center,^{27, 37} and based on the slow ¹⁷O kinetic induction from ¹⁷O-water and the similar ¹⁷O hyperfine to that in the di-μ-oxo model, we assign the ¹⁷O ENDOR signal (¹⁷O_A in Figure 3A) of MnCat to a μ-oxo oxygen in its Mn(III)-Mn(IV) center.

The S₂ state of the OEC has resonance Raman evidence for slow oxygen isotope exchange into a μ-oxo group,²³ and the implication is that there is at least one μ-oxo oxygen in the OEC that can exchange very slowly with solvent. The OEC showed a similar ¹⁷O signal to the slowly exchanging ¹⁷O signal of the ¹⁷O-μ-oxo Mn(III)-Mn(IV) bipyridine and also to the ¹⁷O_A signal in MnCat. The hyperfine coupling from the ¹⁷O signal in the OEC was 11 MHz, which is ~ 15 % less than the hyperfine coupling of the ¹⁷O-μ-oxo in the di-μ-oxo Mn(III)-Mn(IV) bipyridyl complex. A simple explanation for the smaller coupling in the OEC could be that the spin in the tetranuclear Mn center of the OEC is delocalized beyond one di-μ-oxo bridged Mn(III)-Mn(IV) pair.^{58, 59} The tetranuclear center of the OEC is less well defined at present than the bi-nuclear center of Mn(III)-Mn(IV) bipyridine, and so we can not be as confident in our assignment of our ¹⁷O ENDOR signal from the OEC. However, it is extremely tempting to suggest that the ¹⁷O ENDOR signal of the OEC may be from a μ-oxo oxygen within the OEC, in particular, to the μ-oxo oxygen between the distal Mn(III) (Mn No. 4) and its adjacent Mn(IV) partner (Mn No. 2),^{9, 57} which is the oxygen postulated to originate from a “slow exchangeable water” (See Fig. 13 of Kulik et al.⁵⁸).

Beyond the obvious presence of high-valent Mn in all of these systems, their common aspect is μ-oxo oxygen. μ-oxo oxygen mediates via covalent superexchange the antiferromagnetic coupling between paramagnetic metals,⁶⁰ and the detailed electronic nature of antiferromagnetic superexchange is critical to redox/catalytic properties.⁶¹ Broken symmetry DFT has been used to predict spin density at μ-oxo oxygens and pathways for superexchange. Hyperfine couplings for ⁵⁵Mn, ¹⁴N, and ¹H, have been calculated, but not for ¹⁷O.^62–65 Given the central importance of μ-oxo oxygens to all these high-valent multi-manganese complexes, especially the OEC, prediction of the μ-oxo ¹⁷O hyperfine coupling would be a sensitive test of the capabilities of broken symmetry DFT.

The antiferromagnetically coupled Fe(III)-Fe(IV) center of ribonucleotide reductase (RNR) has been the subject of considerable previous ¹⁷O ENDOR.^{46, 66} The μ-oxo ¹⁷O hyperfine tensor in RNR was found to be highly anisotropic (¹⁷A_xx, ¹⁷A_yy, ¹⁷A_zz = 0, 23.5, 23.5 MHz). The μ-oxo ¹⁷O isotropic hyperfine coupling of RNR is 15–16 MHz,⁴⁴ comparable to the isotropic coupling we estimate from our μ-oxo ¹⁷O findings. There was additional hyperfine coupling to a terminal ¹⁷O-water of the Fe(III) with an isotropic component about double that of the μ-oxo, and the μ-oxo ¹⁷O of RNR was distinguished kinetically from the terminal ¹⁷O-water through rapid freeze quench methods to follow rapid incorporation from ¹⁷O₂. The RNR system has advantages over the multi-Mn system that lead to better ENDOR signal to noise and resolution. Due to absence large ⁵⁵Mn nuclear hyperfine couplings, the RNR EPR signal at Q-band is only ~0.1 T wide⁶⁷ versus 1.2–1.8 T wide for di-Mn(III)-Mn(IV), accounting for greater EPR-ENDOR sensitivity for RNR. The RNR system has sufficient g-anisotropy for good angle selection of ¹⁷O ENDOR hyperfine anisotropy; this is not the case for di-Mn(III)-Mn(IV). The μ-oxo couplings of RNR should be larger than those in di-Mn(III)-Mn(IV) systems because Fe tends to be more covalent than Mn and because the S = 5/2 ferric ion has a spin-containing d(x²-y²) orbital directed for σ bonding toward the oxygen 2s and 2p σ-bonding orbital.²²

In analyzing the ¹⁷O ENDOR information, we have simply provided the ENDOR frequency and hyperfine coupling (Eq. 2a) from the peak of the ENDOR feature assigned to ¹⁷O in Figures 3 and 5. Without additional information, we would take this peak frequency as approximately from an isotropic coupling of ~11–13 MHz magnitude. There should, however, be anisotropy in the μ-oxo ¹⁷O hyperfine coupling. A starting point for theoretically estimating hyperfine anisotropy is to calculate the dipolar coupling of the μ-oxo oxygen with electron spin on its Mn(III) and Mn(IV) partners. As explained in the Supporting Information, we applied to μ-oxo ¹⁷O the extended point-dipole model originally developed for predicting dipolar hyperfine interactions of protons near the Mn(III)-Mn(IV) with both metal spins,^{40, 54, 62} and Figure 8S in the Supporting Information provides a simulation⁶⁸ of the μ-oxo ¹⁷O ENDOR feature broadened by hyperfine anisotropy. A goal of future advanced EPR studies of ¹⁷O in the OEC and models for it should be to resolve this hyperfine anisotropy.

¹⁷O ENDOR Assigned to the Terminal Water Ligand on Mn(III) of MnCat

The more weakly coupled ¹⁷O_B inset to Figure 3B with hyperfine coupling (per Eq. 2a) of about 3.8 MHz became obvious from the MnCat sample that had been exchanged only one minute with ¹⁷O-water before freezing. Previous ESEEM^18,69 has provided a hyperfine coupling of about 2 MHz for a ¹⁷O-water sharing π–bonding electrons with low-spin ferric heme in an S = ½ system. Because of the +2 projection factor for Mn(III), the hyperfine coupling for π–bonded ¹⁷O-water on Mn(III) of the Mn(III)-Mn(IV) pair would be about 4 MHz, comparable to that which is obtained here. The quadrupole tensor of ¹⁷O for water ligands of aqua-Mn(II)⁷⁰ and aqua-vanadyl complexes,⁷¹ has two large components (−¹⁷P_xx) = ¹⁷P_yy = ~0.33 MHz and a third, ¹⁷P_zz ~ 0. The expected ¹⁷O-water quadrupole splittings in the ENDOR expressions of Eq. 2 will be |3 ¹⁷P_xx| ~ |3 ¹⁷P_yy| ≈ 1 MHz, comparable with the poorly resolved 1.3 MHz splitting of the shoulders marked in the inset to Figure 3B. We assign the peak feature labeled ¹⁷O_B to terminal ¹⁷O-water on the Mn(III) of the Mn(III)-Mn(IV) pair because there is ENDOR evidence for ¹⁷O with hyperfine coupling and possibly quadrupole couplings consistent with water, because there is a terminal water on Mn in the crystal structure of MnCat, and because there are also proton and deuteron ENDOR data consistent with the protons of that very same water. Further, as discussed below, the terminal water, similar in size to the superoxide substrate of MnCat, would be expected to diffuse into the coordination sphere of the metal center in subsecond times, not hours, and the ¹⁷O_B feature assigned to terminal water does rapidly appear. We point out that a water σ-bonded along a Jahn-Teller distorted axis of Mn(III) might in principle be expected to have larger hyperfine coupling than water π-bonded to Mn(III) or Mn(IV), but we do not observe a large coupling to a rapidly incorporated ¹⁷O.

Exchangeable Proton and Deuteron ENDOR Assigned to the Terminal Water Ligand on Mn(III) of MnCat

The largest proton coupling of 17.2 MHz (singly starred in Figure 4) is comparable to the largest coupling of 17.6 MHz observed from terminal water on Mn(III) from the (2-OH-3,5-Cl₂-SALPN)₂ Mn(III)-Mn(IV) complex.⁵⁴ In the crystal structure of the di-Mn active site in MnCat (Figure 1) there is a carboxylate oxygen of Glu178 within a 2.5 Å hydrogen-bonding distance of the terminal water oxygen with a Mn-O-H-O bond angle of 120°. We have positioned a hydrogen-bonded proton along the water-oxygen-to-carboxylate-oxygen direction at 1.0 Å from the water oxygen. The geometric parameters for that water proton are provided in the Supporting Infomration (See Scheme 1S and pages S11 and S12 in the supporting Information.) The dipolar coupling to this proton is ¹A_xxdip, ¹A_yydip, ¹A_zzdip = −8.6, −6.6, +15.2 MHz. In addition to the dipolar coupling of ¹A_zzdip = 15.2 MHz, the total hyperfine coupling of ¹A_zz = 17.2 MHz of the starred features in Figure 4 would require a small +2.0 MHz isotropic component, comparable to that suggested by Randall et al.⁵⁴ We assign the proton that gives rise to the observed coupling of 17.2 MHz in Figure 4 to the water that also gives the ¹⁷O coupling of 3.8 MHz for ¹⁷O_B. The predicted smaller perpendicular dipolar components, ¹A_xxdip, ¹A_yydip, which are 6–9 MHz in magnitude, would be largely buried here in the proton ENDOR spectra of Figure 4 under non-exchangeable proton ENDOR signals, but the deuteron ENDOR shown in the inset to Figure 4 does have intensity in the 0.9–1.4 MHz region that may correspond to the 6–9 MHz proton region. Water ligated to Mn(III) may have a second proton located further from the Mn(III) than the one with the coupling of 17.2 MHz. Dipolar calculations performed on a second water proton a few tenths of an Å further from the Mn(III) predicted a smaller maximal proton dipolar coupling in the 12–14 MHz range, consistent with the outlying doubly starred proton hyperfine signals having a splitting of 12.2 MHz in Figure 4.

Exchange of Water into the Manganese Active Sites

Q-band ENDOR and X-band EPR showed that the solvent-derived ¹⁷O ligands appeared in the di-μ-oxo core of Mn(III)-Mn(IV) bipyridyl²² within tens of minutes and in the di-μ-oxo core of MnCat within several hours. Q-band ¹⁷O ENDOR and Resonance-Raman-detectable ¹⁸O at a μ-oxo site²³ within PSII occurred on a similar time scale. If the μ-oxo ¹⁷O observed by ENDOR of PSII is the same μ-oxo that Resonance Raman has resolved after slow exchange,²³ then this oxygen would not be catalytically relevant in the sense that it rapidly appears in the O₂ product, although it is relevant to the structure of the catalytic center. (It is likely that the bridge opening to allow oxygen exchange requires a proton from a nearby base,²⁴ such as a nearby arginine in the OEC.)

MnCat functions with a time for superoxide turnover of 5 μs, close to the diffusion limit.³⁰ Superoxide, and very likely the similar-sized water, can diffuse into the active site very easily, so that diffusion of water into the interior of the MnCat hexamer,²⁷ as opposed to exchange into the μ-oxo Mn(III)-Mn(IV) core, is unlikely to be an impediment to oxygen isotope exchange for MnCat. The evidence for terminal water ligation from H₂¹⁷O samples frozen within a minute of the initiation of water exchange, is consistent with such a rapid exchange, which indeed may even occur in a much shorter time than a minute.

The large ENDOR-resolved proton coupling in MnCat (¹A_zz > 15 MHz) indicating ligation of a terminal water to Mn(III) of the di-μ-oxo Mn(III)-Mn(IV) pair in MnCat has not yet been observed in PSII. We would interpret the hyperfine couplings of so far unresolved exchangeable proton/deuterons in the OEC derived from ENDOR and ESEEM,^{72, 73} as being too small to be those of water protons on a Mn(III) of a Mn(III)-Mn(IV) antiferromagnetically coupled pair. We cannot at this point exclude hydrogen bonding to a bridging μ-oxo between Mn and Ca as a candidate for ESEEM-observable hydrogen bonds in PSII because to date there is not a Mn-Ca model to test this hypothesis.

Conclusion

Although there was previous ENDOR evidence for exchange of ¹⁷O from ¹⁷O-water into the μ-oxo ligands of the Mn(III)-Mn(IV) bipyridine model complex,^{21, 22, 24} the spectroscopic signatures had not been explored for ¹⁷O exchanged from ¹⁷O-water into a protein active site having the potential to exchange both μ-oxo and terminal water oxygens. MnCat, di-μ-oxo Mn(III)-Mn(IV) bipyridine, and the S₂ signal of the OEC have now all provided a similar ¹⁷O ENDOR signal that arose after slow exchange with ¹⁷O-water. This signal is assigned to a μ-oxo oxygen that joins a Mn(III)-Mn(IV) antiferromagnetically coupled pair. This μ-oxo oxygen, an important structural aspect of multi-Mn centers, exchanged on the scale of tens of minutes into the Mn(III)-Mn(IV) bipyridine complex and on the time scale of hours into the μ-oxo Mn(III)-Mn(IV) centers of MnCat and the OEC. Thus, the rapid water binding sites for the OEC are most likely to be terminal, or even more distant, water binding sites. As judged by the small magnitude of exchangeable ¹H and ²H hyperfine couplings for the OEC^72,73 when compared to the > 15 MHz exchangeable proton coupling from the assigned terminal water ligand of Mn(III) in Mn(III)-Mn(IV) MnCat, these sites in the OEC are not of a terminal water to the Mn(III) of a Mn(III)-Mn(IV) pair. Hydrogen bonding to a μ-oxo ligand between Mn and Ca cannot be excluded.

For MnCat, ENDOR evidence was obtained for terminal water exchange with H₂¹⁷O on a minute (or less) time scale. ¹⁷O hyperfine and possible ¹⁷O quadrupole ENDOR signals, which differed significantly from ¹⁷O-μ-oxo ENDOR signals, indicated terminal water. In addition there were large exchangeable proton couplings consistent with the dipole interaction of a water ligand proton with a near Mn(III) and a more distant Mn(IV); exchangeable deuteron signals, although less well resolved, were also consistent with the deuterium on the same terminal water.

In Scheme 1 we succinctly present the outcomes reported in this article. Scheme 1 summarizes the time span for the appearance of ¹⁷O-ENDOR peak positions from MnCat and the OEC and for our assignment of the physical location of these ¹⁷O within the manganese centers of MnCat and the OEC. The assignment of exchangeable proton/deuteron ENDOR to the exchangeable water on the Mn(III) of MnCat is also noted.

Scheme 1.

shows the following information: a) The time course for H₂¹⁷O binding and ¹⁷O-migration within the limits of the present experiments. b) The location of the ¹⁷O peaks as the time course of the experiment progresses., including the labels ¹⁷O_A for μ-oxo and ¹⁷O_B from a ¹⁷O-water ligand. c) The location of the exchangeable H on terminal water ligand of Mn(III) in MnCat. is shown. d) The labels of Mn(4) and Mn(3) are indicated per Sproviero et al. ⁵⁵