Iron-Blocking the High-Affinity Mn-Binding Site in Photosystem II Facilitates Identification of the Type of Hydrogen Bond Participating in Proton-Coupled Electron Transport via Yz

Incubation of Mn-depleted PSII membranes [PSII(-Mn)] with Fe(II) is accompanied by the blocking of Y_Z^. at the high-affinity Mn-binding site to exogenous electron donors [Semin et al. (2002) Biochemistry 41, 5854-5864] and a shift of the pK_app of the hydrogen bond partner for Y_Z (base B) from 7.1 to 6.1 [Semin, B. K., and Seibert, M. (2004) Biochemistry 43, 6772-6782]. Here we calculate activation energies (E _a) for Y_Z^. reduction in PSII(-Mn) and Fe-blocked PSII(-Mn) samples [PSII(-Mn, +Fe)] from temperature dependencies of the rate constants of the fast and slow components of the flash-probe fluorescence decay kinetics. At pH < pK_app (e.g., 5.5), the decays are fit with one (fast) component in both types of samples, and E_a is equal to 42.2 ± 2.9 kJ/mol in PSII(-Mn) and 46.4 ± 3.3 kJ/mol in PSII(-Mn, +Fe) membranes. At pH > pK_app, the decay kinetics exhibit an additional slow component in PSII(-Mn, +Fe) membranes (E_a = 36.1 ± 7.5 kJ/mol), which is much lower than the E_a of the corresponding component observed for Y_Z^. reduction in PSII(-Mn) samples (48.1 ± 1.7 kJ/mol). We suggest that the above difference results from the formation of a strong low barrier hydrogen bond (LBHB) between Y_Z and base B in PSII(-Mn, +Fe) samples. To confirm this, Fe-blocking was performed in D₂O to insert D⁺, which has an energetic barrier distinct from H⁺, into the LBHB. Measurement of the pH effects on the rates of Y_Z^. reduction in PSII(-Mn, +Fe) samples blocked in D₂O shows a shift of the pK_app from 6.1 to 7.6, and an increase in the E_a of the slow component. This approach was also used to measure the stability of the Y_Z^. EPR signal at various temperatures in both kinds of membranes. In PSII-(-Mn) membranes, the freeze-trapped Y_Z^. radical is stable below 190 K, but half of the Y_Z^. EPR signal disappears after a 1-min incubation when the sample is warmed to 253 K. In PSII(-Mn, +Fe) samples, the trapped Y_Z^. radical is unstable at a much lower temperature (77 K), However, the insertion of D ⁺ into the hydrogen bond between Y_Z and base B during the blocking process increases the temperature stability of the Y_Z ^. EPR signal at 77 K. Again, these results indicate that Fe-blocking involves Y_Z in the formation of a LBHB, which in turn is consistent with the suggested existence of a LBHB between Y_Z and base B in intact PSII membranes [Zhang, C., and Styring, S. (2003) Biochemistry 42, 8066-8076].