Calcium induces binding and formation of a spin-coupled dimanganese(II,II) center in the apo-water oxidation complex of photosystem II as precursor to the functional tetra-Mn/Ca cluster

TWO new intermediates are described which form in the dark as precursors to the light-induced assembly of the photosynthetic water oxidation complex (WOC) from the inorganic components. Mn2+ binds to the apo-WOC-PSII protein in the absence of calcium at a high-affinity site. By using a hydrophobic chelator to remove Mn2+ and Ca2+ from the WOC and nonspecific Fe3+, a new EPR signal becomes visible upon binding of Mn2+ to this site, characterized by six-line Mn-55 hyperfine structure (Delta H-pp = 96 +/- 1 G) and effective g = 8.3. These features indicate a high-spin electronic ground state (S = 5/2) for Mn2+ and a strong ligand field with large anisotropy, This signal is eliminated if excess Ca2+ or Mg2+ is present. A second Mn2+ EPR signal forms in place of this signal upon addition of Ca2+ in the dark. The yield of this Ca-induced Mn signal is optimum at a ratio of 2 Mn/PSII, and saturates with increasing [Ca2+] greater than or equal to 8 mM, exhibiting a calcium dissociation constant of K-D = 1.4 mM. The EPR signal of the Ca-induced Mn center at 25 K is asymmetric with major g value of approximate to 2.05 (Delta H-pp = 380 G) and a shoulder near g approximate to 3.1. It also exhibits resolved Mn-55 hyperfine splitting with separation Delta H-pp = 42-45 G. These spectral features are diagnostic of a variety of weakly interacting Mn-2(II,II) pairs with electronic spins that are magnetic dipolar coupled in the range of intermanganese separations 4.1 +/- 0.4 Angstrom, and commonly associated with one or two carboxylate bridges. The calcium requirement for induction of the Mn-2(II,II) signal matches the value observed for steady-state O-2 evolution (Michaelis constant, K-M approximate to 1.4 mM), and for light-induced assembly of the WOC by photoactivation. The Ca-induced Mn-2(II,II) center is a more efficient electron donor to the photooxidized tyrosine radical, Tyr(Z)(+), than is the mononuclear Mn center present in the absence of Ca2+. The Ca-induced Mn-2(II,II) signal serves as a precursor for photoactivation of the functional WOC and is abolished by the presence of Mg2+. Formation of the Mn-2(II,II) EPR signal by addition of Ca2+ correlates with reduction of flash-induced catalase activity, indicating that calcium modulates the accessibility or reactivity of the Mn-2(II,II) core with H2O2. We propose that calcium organizes the binding site for Mn ions in the apo-WOC protein and may even interact directly with the Mn-2(II,II) pair via solvent or protein-derived bridging ligands.