Is Mn-Bound Substrate Water Protonated in the S(2) State of Photosystem II?

Abstract

In spite of great progress in resolving the geometric structure of the water-splitting Mn(4)O(x)Ca cluster in photosystem II, the binding sites and modes of the two substrate water molecules are still insufficiently characterized. While time-resolved membrane-inlet mass spectrometry measurements indicate that both substrate water molecules are bound to the oxygen-evolving complex (OEC) in the S(2) and S(3) states (Hendry and Wydrzynski in Biochemistry 41:13328-13334, 2002), it is not known (1) if they are both Mn-bound, (2) if they are terminal or bridging ligands, and (3) in what protonation state they are bound in the different oxidation states S(i) (i = 0, 1, 2, 3, 4) of the OEC. By employing (17)O hyperfine sublevel correlation (HYSCORE) spectroscopy we recently demonstrated that in the S(2) state there is only one (type of) Mn-bound oxygen that is water exchangeable. We therefore tentatively identified this oxygen as one substrate 'water' molecule, and on the basis of the finding that it has a hyperfine interaction of about 10 MHz with the electron spin of the Mn(4)O(x)Ca cluster, we suggest that it is bound as a Mn-O-Mn bridge within a bis-mu(2) oxo-bridged unit (Su et al. in J Am Chem Soc 130:786-787, 2008). Employing pulse electron paramagnetic resonance, (1)H/(2)H Mims electron-nuclear double resonance and (2)H-HYSCORE spectroscopies together with (1)H/(2)H-exchange here, we test this hypothesis by probing the protonation state of this exchangeable oxygen. We conclude that this oxygen is fully deprotonated. This result is discussed in the light of earlier reports in the literature.

Fig. 1

Light-minus-dark ESE-detected field-swept X-band EPR spectrum of the S ₂ state in PSII membrane fragments at 4.2 K. The sample was highly enriched (>95%) in ²H₂O and had a Chl concentration of about 20–30 mg/ml. The used pulse sequence is a π/2–τ–π pulse, where π/2 = 12 ns, τ = 128 ns, and shots repetition time (SRT) = 5 ms with microwave frequency of 9.70 GHz. The displayed signal represents the average of four scans. The absorption of the Tyr_D^· radical was removed for the presentation. The vertical bar and the arrow indicate the field positions where the Mims ENDOR and ESEEM/HYSCORE were measured in Figs. 2, 3 and 4, respectively

Fig. 2

¹H/²H Mims ENDOR of concentrated PSII membranes from spinach suspended either in ²H₂O-enriched (black) or in ¹H₂O-buffer (gray). The data were recorded with a pulse sequence π/2–τ–π/2–t _rf–π/2–τ-echo, where π/2 = 200 ns, τ = 420 ns, the length of rf pulse t _rf was 25 μs in both X- and Q-band measurements. The Larmor frequencies of ²H in X-band (B ₀ = 335 mT, 9.70 GHz) and Q-band (B ₀ = 1220 mT, 33.93 GHz) are 2.19 and 7.974 MHz, respectively. For ¹H, it is 14.26 MHz (B ₀ = 335 mT) in X-band. The inset spectrum was measured at Q-band (²H₂O-enriched sample). The arrows indicate the positions of ¹H-signals from exchangeable protons

Fig. 3

2D-3P ESEEM of concentrated PSII membranes from spinach suspended in ²H₂O-enriched buffer. The spectra were recorded with the pulse sequence π/2–τ–π/2–T–π/2–τ-echo, where π/2 = 24 ns and τ was varied from 108 to 888 ns in 20 ns steps. T was varied from 48 to 6192 ns in 24 ns steps. Four-phase cycling with 50 shots/point and a shot repetition time of 5 ms was employed. The magnetic field was set to B ₀ = 355 mT. The Larmor frequencies of ¹H and ²H are 15.115 and 2.32 MHz, respectively. After Fourier transformation, the projections on the top and on the right show the 1D spectrum in the frequency domain, and the oscillation of the signal amplitude as a function of τ, respectively

Fig. 4

X-band ²H-HYSCORE spectrum of concentrated PSII membranes from spinach suspended in ²H₂O-enriched buffer. The spectrum was recorded with a pulse sequence π/2–τ–π/2–t ₁–π–t ₂–π/2–τ-echo with π/2 = 24 ns. The times t ₁ and t ₂ were varied from 60 to 6,720 ns in 24 ns steps. Four-phase cycling with 50 shots/point and a shot repetition time of 5 ms was employed. Other parameters: B ₀ = 355 mT, τ = 256 ns and the microwave frequency was 9.70 GHz

Fig. 5

Proposed substrate ‘water’ binding modes and sites at the S ₂ state of the Mn₄O_xCa. W _s and W _f represent the slow- and the fast-exchanging substrate ‘water’ molecules as determined by time-resolved membrane-inlet mass spectrometry [16, 44]. The four Mn ions are denoted as A, B, C, D, while the oxygen ions are represented by dark spheres. The structural model employed is based on single-crystal EXAFS spectroscopy [2] and theoretical calculations [61]