| Abstract |
Lanthanide ions compete and replace calcium in its binding sites in PSII. Lanthanide substituted PSII membranes retain all three extrinsic polypeptides 17, 23 and 33 kDa, and the manganese complex. The enzyme which results with lanthanide treatment does not evolve oxygen. In the present study, EPR spectroscopy, thermoluminescence, and optical spectroscopy were used to probe at the oxidizing and reducing site of PSII, and the electron transfer properties of lanthanide treated and calcium depleted HM-cores. In lanthanide treated PSII membranes, the electron tranfer rate from the manganese complex to tyrosine YZi is slowed, allowing accumulation of YZi spins. At pH 7,5, the tyrosine YZi is fully developed with high quantum efficiency, compatible to an inhibited system such as Tris-PSII, which lacks the manganese cluster, and the extrinsic 17, 23, and 33 kDa polypeptides. It was demonstrated by kinetic EPR experiments, using Ln-PSII, that neither the extrinsic polypeptides (17, 23, and 33 kDa), nor the manganese complex block the accessibility of YZi to exogenous reductants such as benzidine. In addition, it was shown that in the presence of the native manganese complex, exogenous Mn2+ has no access to tyrosine YZi. At least one lanthanide ion binding site is close to tyrosine YZ as revealed by the power saturation properties of YZi in the presence of paramagnetic lanthanides. When diamagnetic ions occupy the calcium binding site(s), there was not a magnetic interaction between the tyrosine YZi and the (Mn)4 cluster. Therefore, in the presence of lanthanide ions, the modified manganese cluster is EPR silent, perhaps in a diamagnetic conformation. In the lanthanide treated PSII membranes the rebinding of 35Cl- ions are prevented. Chloride ions are required for oxygen evolution since they participate in the protolytic chemistry. Thus, an explanation can be given for the lack of oxygen evolution in Ln-PSII membranes. Electron Paramagnetic Resonance spectroscopy at low temperatures, He (l), and thermoluminescence experiments demonstrated that in Ln-PSII membranes, the manganese cluster does not proceed to higher oxidation states. Also, upon NO addition, neither the non-heam Fe2+-NO adduct, nor the Mn(II)-Mn(III), attributed to S0 and S-1 state of the manganese complex are formed. Reconstitution of the extrinsic 17 and 23 kDa polypeptides restored the EPR signal, g=1.84 (Fe2+-QA-). The observed effects on the reducing site are an example of a long range communication of changes introduced at the oxidizing site, and through the protein matrix, extended to the reducing site. At pH 6,0, in Ln-PSII systems, the tyrosine YZ is the intermediate electron carrier between the Mn cluster and the light oxidazable chlorophyll P680+. As detected by kinetic EPR, tyrosine YZi spins are accumulated slowly, in a low quantum yield, in a manner analogous to (2 N) NaCl treated and Ca2+ depleted PSII membranes. Time resolved optical spectroscopy showed that tyrosine YZi in lanthanide treated HM-core, at pH 6,0, reduced P680+, with half life time in the order of microseconds (μsec) as in NaCl/EGTA and Ca2+ depleted systems. It was observed that when a (2 Ν) NaCl treated PSII, at pH 7,5 was exposed to reductants such as HQ, and NH2OH, the (Mn)4 cluster was retained intact at the oxygen evolving complex. On the contrary, when a lanthanide treated PSII sample, at pH 7,5, was exposed to the above reductants the manganese cluster was reduced and released, while incubation with Ca2+ did not protect the manganese cluster. These results support that calcium maintains the structural integrity of the oxygen evolving complex.
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