Effective binding of Tb3+ and La3+ cations on the donor side of Mn-depleted photosystem II.

The interaction of Tb3+ and La3+ cations with different photosystem II (PSII) membranes (intact PSII, Ca-depleted PSII (PSII[-Ca]) and Mn-depleted PSII (PSII[-Mn]) membranes) was studied. Although both lanthanide cations (Ln3+) interact only with Ca2+-binding site of oxygen-evolving complex (OEC) in PSII and PSII(-Ca) membranes, we found that in PSII(-Mn) membranes both Ln3+ ions tightly bind to another site localized on the oxidizing side of PSII. Binding of Ln3+ cations to this site is not protected by Ca2+ and is accompanied by very effective inhibition of Mn2+ oxidation at the high-affinity (HA) Mn-binding site ([Mn2+ + H2O2] couple was used as a donor of electrons). The values of the constant for inhibition of electron transport Ki are equal to 2.10 ± 0.03 µM for Tb3+ and 8.3 ± 0.4 µM for La3+, whereas OEC inhibition constant in the native PSII membranes is 323 ± 7 µM for Tb3+. The value of Ki for Tb3+ corresponds to Ki for Mn2+ cations in the reaction of diphenylcarbazide oxidation via HA site (1.5 µM) presented in the literature. Our results suggest that Ln3+ cations bind to the HA Mn-binding site in PSII(-Mn) membranes like Mn2+ or Fe2+ cations. Taking into account the fact that Mn2+ and Fe2+ cations bind to the HA site as trivalent cations after light-induced oxidation and the fact that Mn cation bound to the HA site (Mn4) is also in trivalent state, we can suggest that valency may be important for the interaction of Ln3+ with the HA site.

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 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(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].