Calcium Binding Studies of Photosystem II Using a Calcium-Selective Electrode

Abstract

The identification of Ca2+ as a cofactor in photosynthetic O2 evolution has encouraged research into the role of Ca2+ in photosystem II (PSII). Previous methods used to identify the number of binding sites and their affinities were not able to measure Ca2+ binding at thermodynamic equilibrium. We introduce the use of a Ca2(+)-selective electrode to study equilibrium binding of Ca2+ to PSII. The number and affinities of binding sites were determined via Scatchard analysis on a series of PSII membrane preparations progressively depleted of the extrinsic polypeptides and Mn. Untreated PSII membranes bound approximately 4 Ca2+ per PSII with high affinity (K = 1.8 microM) and a larger number of Ca2+ with lower affinity. The high-affinity sites are assigned to divalent cation-binding sites on the light-harvesting complex II that are involved in membrane stacking, and the lower-affinity sites are attributed to nonspecific surface-binding sites. These sites were also observed in all of the extrinsic polypeptide- and Mn-depleted preparations. Depletion of the extrinsic polypeptides and/or Mn exposed additional very high-affinity Ca2(+)-binding sites which were not in equilibrium with free Ca2+ in untreated PSII, owing to the diffusion barrier created by the extrinsic polypeptides. Ca2(+)-depleted PSII membranes lacking the 23 and 17 kDa extrinsic proteins bound an additional 2.5 Ca2+ per PSII with K = 0.15 microM. This number of very high-affinity Ca2(+)-binding sites agrees with the previous work of Cheniae and co-workers [Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721-724, Kluwer, Dordrecht, The Netherlands] whose procedure for Ca2+ depletion was used. Further depletion of the 33 kDa extrinsic protein yielded a sample that bound only 0.7 very high-affinity Ca2+ per PSII with K = 0.19 microM. The loss of 2 very high-affinity Ca2(+)-binding sites upon depletion of the 33 kDa extrinsic protein could be due to a structural change of the O2-evolving complex which lost 2-3 of the 4 Mn ions in this sample. Finally, PSII membranes depleted of Mn and the 33, 23, and 17 kDa extrinsic proteins bound approximately 4 very high-affinity Ca2+ per PSII with K = 0.08 microM. These sites are assigned to Ca2+ binding to the vacant Mn sites.

Structure and Orientation of the Mn4Ca Cluster in Plant Photosystem II Membranes Studied by Polarized Range-extended X-ray Absorption Spectroscopy

“…Calcium was included in the fit combination because it has been implicated as a structural element of the OEC through O 2 evolution activity (19,45,(62)(63)(64)(65)(66), EPR (20), and calcium-and strontium-EXAFS experiments (22,23). In conventional EXAFS experiments it was noticed previously that the addition of the manganese-calcium vector to the manganese-manganese long interaction improves fit qualities.…”

Dark-Stimulated Calcium Ion Fluxes in the Chloroplast Stroma and Cytosol

“…When photosynthetic electron transport is allowed to proceed, Ca 2ϩ that is taken up during the day does not accumulate in the stroma as free Ca 2ϩ ; rather, it is either transported into other compartments (e.g., the thylakoid) or bound (Kreimer et al, 1987). It has been reported that Ca 2ϩ performs vital functions in the thylakoid (Becker et al, 1985;Miller and Brudvig, 1989;Grove and Brudvig, 1998). A Ca 2ϩ /H ϩ antiporter has been characterized from thylakoids (Ettinger et al, 1999), and it is attractive to consider the possibility that this antiporter transports Ca 2ϩ into the thylakoid, driven by the proton electrochemical gradient generated by photosynthetic electron transport.…”

“…On the other hand, within the thylakoid, functional assembly of photosystem II (PSII) and the oxygen-evolving complex requires proper assembly of polypeptides and cofactors in a process that is light and Ca 2 ϩ dependent (Becker et al, 1985;Miller and Brudvig, 1989;Grove and Brudvig, 1998). This Ca 2 ϩ dependence is relevant not only to the initial assembly of the PSII/oxygen-evolving complex and to the repair of PSII reaction centers that have been damaged by photoinhibition during a normal day (Mattoo et al, 1989) but also to the mechanism of photosynthetic oxygen evolution (Yocum, 1991).…”

The PSII calcium site revisited

“…A second Ca 2+ atom was found to be tightly bound to antenna proteins (Han and Katoh 1993), probably CP29 (Jegerschö ld et al 2000). Binding of Ca 2+ has also been examined under static (dark) conditions with an ion-specific electrode (Grove and Brudvig 1998 (Vrettos et al 2001a;van Gorkom and Yocum 2005) with regard to the ability of inhibitory metals to occupy the Ca 2+ site. The lanthanides that have been tested are all effective at low (50-100 lM) concentrations, and tend to produce inhibitions that are difficult to reverse (Ghanotakis et al 1985;Bakou et al 1992).…”