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 a p p of the hydrogen bond partner for Yz (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 a p p , 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 D 2 O 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 D 2 O shows a shift of the pK a p p 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 Yz 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].