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(center dot) 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(center dot) 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(center dot) 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(center dot) 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(center dot) EPR signal at various temperatures in both kinds of membranes. In PSII(-Mn) membranes, the freeze-trapped Y-Z(center dot) radical is stable below 190 K, but half of the Y-Z(center dot) 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(center dot) 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(center dot) 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].