Long lived intermediate metal site structure upon binding of cadmium to plastocyanin.

Perturbed angular correlation of gamma-rays (PAC) spectroscopy of cadmium substituted plastocyanin shows one dominant metal site configuration at pH 7.5. Lowering the pH to 4.8 a fraction of the molecules undergoes structural change and loses the bound cadmium ion. At pH 4.4 all plastocyanin is in the apo-form. Increasing the pH back to neutral pH values two distinct metal site coordination geometries were observed. One of the two signals is the same as that found initially at pH 7.5; the other form is stable for hours at 1 degrees C, indicating the existence of a long lived intermediate metal site structure. The cadmium ion is surrounded by the same ligands (His37, Cys84, His87 and Met92) in both forms, however the metal center in the long lived intermediate metal site structure can be best described by a larger His-metal-His angle.

Fast cadmium inhibition of photosynthesis in cyanobacteria in vivo and in vitro studies using perturbed angular correlation of gamma-rays.

The effect of cadmium on the photosynthetic activity of Synechocystis PCC 6803 was monitored in this study. The oxygen evolving capacity of Synechocystis treated with 40 muM CdCl(2) was depressed to 10% of the maximum in 15 min, indicating that Cd(2+) penetrated rapidly into the cells and blocked the photosynthetic activity. However, neither photosystem II (PSII) nor photosystem I (PSI) activity showed a significant short-term decrease which would explain this fast decrease in the whole-chain electron transport. Thermoluminescence measurements have shown that the charge separation and stabilization in PSII remains essentially unchanged during the first few hours following the Cd(2+) treatment. The electron flow through PSI was monitored by following the redox changes of the P700 reaction centers of PSI. Alterations in the oxidation kinetics of P700 in the Cd(2+)-treated cells indicated that Cd(2+) treatment might affect the available electron acceptor pool of P700, including the CO(2) reduction and accumulation in the cells. Perturbed angular correlation of gamma-rays (PAC) using the radioactive (111m)Cd isotope was used to follow the Cd(2+) uptake at a molecular level. The most plausible interpretation of the PAC data is that Cd(2+) is taken up by one or more Zn proteins replacing Zn(2+) in Synechocystis PCC 6803. Using the radioactive (109)Cd isotope, a protein of approximately 30 kDa that binds Cd(2+) could be observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results indicate that Cd(2+) might inactivate different metal-containing enzymes, including carbonic anhydrase, by replacing the zinc ion, which would explain the rapid and almost full inhibition of the photosynthetic activity in cyanobacteria.

pH-dependent structural change of reduced spinach plastocyanin studied by perturbed angular correlation of gamma-rays and dynamic light scattering.

In this study the pH-dependent structural changes of reduced spinach plastocyanin were investigated using perturbed angular correlation (PAC) of gamma-rays and dynamic light scattering (DLS). PAC data of Ag-substituted plastocyanin indicated that the coordinating ligands are two histidine residues (His37, His87) and a cysteine residue (Cys84) in a planar configuration, whereas the methionine (Met92) found perpendicular to this plane is not a coordinating ligand at neutral pH. Two slightly different conformations with differences in the Cys-metal ion-His angles could be observed with PAC spectroscopy. At pH 5.3 a third coordination geometry appears which can be explained as the absence of the His87 residue and the coordination of Met92 as a ligand. With DLS the aggregation of reduced plastocyanin could be observed below pH 5.3, indicating that not only the metal binding site but also the aggregation properties of the protein change upon pH reduction. Both the structural changes at the metal binding site and the aggregation are shown to be reversible. These results support the hypothesis that the pH of the thylakoid lumen has to remain moderate during steady-state photosynthesis and indicate that low pH induced aggregation of plastocyanin might serve as a regulatory switch for photosynthesis.