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

Abstract

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

Keywords: Mn cluster; OEC; PSII; water oxidizing complex; water-oxidation.

Fig. 1.

(A) The S-state cycle of the OEC. The crystal structure of the manganese tetramer is also shown, indicating the unusual ligation of O5, equidistant between Mn_A4 and Mn_D1 (1). (B) A representative, unified DFT model of the OEC in the S₂ state (10). The oxygen ligands W1 (pink), W2 (cyan), and O4/O5 (green) were assigned as sites exchangeable with solvent water in the S₁ state (17). (C) The effect of ammonia on the CW-EPR (multiline) signal of the S₂ state [11,29] (Fig. S1 and Table S1).

Fig. 2.

(A) X-band three-pulse ESEEM traces measured at the center of the S₂-state multiline signal (Fig. 1, B₀ = 333 mT, microwave frequency = 9.4 GHz). The data represent annealed-minus-dark difference traces collected on ammonia (¹⁴NH₃)-treated ¹⁴N-PSII (black) and (¹⁴NH₃)-treated ¹⁵N-PSII (blue). The traces shown in A were measured with an interpulse spacing τ of 136 ns. Additional data traces using the τ-values 152 ns, 168 ns, and 184 ns are shown in Fig. S2. (B) Fourier transform (FT) of the X-band time domain data. N.Q.L. identifies the nuclear-quadrupole lines caused by the coupling of the OEC with the added ¹⁴N (I = 1). The spectrum shown represents the sum of the FT of the four ESEEM traces measured using different τ-values (136–184 ns) to minimize spectral artifacts. (C) Q-band three-pulse ESEEM traces measured at the center of the S₂-state multiline signal (B₀ = 1.22 T, microwave frequency = 34.0 GHz). The data represent light-minus-dark and annealed-minus-dark difference spectra of native ¹⁴N-PSII (black) and ammonia (¹⁴NH₃)-treated ¹⁴N-PSII (blue) respectively. The time domain data were measured using an interpulse spacing τ of 260 ns. Additional data traces using τ-values of 240 ns and 300 ns are shown in Fig. S2. (D) Corresponding FT of the data traces presented in C. S.Q. and D.Q. identify single-quantum and double-quantum transition lines from the coupling with ¹⁴N-His332. The red dashed lines superimposing the data represent a simulation using the spin Hamiltonian formalism (SI EPR Theory/Simulations, Fig. S2, and Table S2). The label N.Q.L. identifies the quadrupole lines observed in the X-band ¹⁴N-ESEEM spectrum.

Fig. 3.

(A) W-band ¹⁷O-EDNMR spectra of native and ¹⁴NH₃-treated ¹⁴N-PSII samples (17). The black line represents the data; the red dashed line represents the total simulation. Fitted isotropic hyperfine values are listed in Table 1. A complete list of parameters is given in Table S3. The colored traces represent the four components of the fit: the ¹⁴N of D1-His332, blue; the strongly coupled ¹⁷O species, green; the intermediately coupled ¹⁷O species, orange; and the weakly coupled ¹⁷O species, pink. (B) TR-MIMS traces monitoring substrate exchange in the S₂ state at pH 7.6 in the presence of either 100 mM NH₄Cl (red triangles) or 100 mM NaCl (black squares). The lines represent biexponential (³⁴O₂, Left) and monoexponential (³⁶O₂, Right) fits. NH₄Cl: k_f = 52 s⁻¹, k_s = 2 s⁻¹. NaCl: k_f = 38 s⁻¹, k_s = 3 s⁻¹.

Fig. 4.

(Left) Site for NH₃ binding to the OEC poised in the S₂ state. NH₃ displaces W1, a water ligand of the outer Mn_A4 (a Mn^IV ion in the S₂ state), which slightly affects the binding strength of the oxo-bridge O5, which is trans to this position. (Right) O-O bond formation mechanisms consistent with this study (see main text): (I) a nucleophilic attack of O5 by a nearby substrate; (II) an oxo/oxyl radical coupling of O5 and an as yet unidentified additional water marked W_f (possibly W2). Mn, purple; Ca, yellow; N, blue; O, red; and substrate O, green.