Spectroscopic Evidence for Ca²⁺Involvement in the Assembly of the Mn₄Ca Cluster in the Photosynthetic Water-Oxidizing Complex

Abstract

Biogenesis and repair of the inorganic core (Mn 4 CaO x Cl y ), in the water-oxidizing complex of photosystem II (WOC-PSII), occurs through the light-induced (re)assembly of its free elementary ions and the apo-WOC-PSII protein, a reaction known as photoactivation. Herein, we use electron paramagnetic resonance (EPR) spectroscopy to characterize changes in the ligand coordination environment of the first photoactivation intermediate, the photo-oxidized Mn 3+ bound to apo-WOC-PSII. On the basis of the observed changes in electron Zeeman (g eff ), 55 Mn hyperfine (A Z ) interaction, and the EPR transition probabilities, the photogenerated Mn 3+ is shown to exist in two pH-dependent forms, differing in terms of strength and symmetry of their ligand fields. The transition from an EPR-invisible low-pH form to an EPR-active high-pH form occurs by deprotonation of an ionizable ligand bound to Mn 3+ , implicated to be a water molecule: [Mn 3+ (OH 2 In the absence of Ca 2+ , the EPR-active Mn 3+ exhibits a strong pH dependence (pH ∼6.5-9) of its ligand-field symmetry (rhombicity ∆δ ) 10%, derived from g eff ) and A Z (∆A Z ) 22%), attributable to a protein conformational change. Binding of Ca 2+ to its effector site eliminates this pH dependence and locks both g eff and A Z at values observed in the absence of Ca 2+ at alkaline pH. Thus, Ca 2+ directly controls the coordination environment and binds close to the highaffinity Mn 3+ , probably sharing a bridging ligand. This Ca 2+ effect and the pH-induced changes are consistent with the ionization of the bridging water molecule, predicting that [ A single tight-binding Ca 2+ ion is essential for photosynthethic O 2 evolution activity in ViVo (1-5). Calcium is located within the water-oxidizing complex of photosystem II (PSII-WOC), 1 comprised of an oxo/aquo-bridged inorganic core whose stoichiometry, Mn 4 CaO x , is based on several lines of evidence, including X-ray absorption (6-9), electron paramagnetic resonance (EPR) spectroscopy (10,11), and the recent X-ray diffraction (XRD) data (12, 13). Mn-, Sr-, and Ca-extended X-ray absorption fine structure (EXAFS) spectroscopies accurately place the calcium effector site at 3.3-3.5 Å from Mn atoms. However, the relative position of Ca 2+ within the Mn 4 O x cluster differs according to which type of data is emphasized and the assumptions of the models used to interpret the raw data. Spectroscopic and XRD data have constrained the possible core topologies to only a few types, with the structures given in Chart 1 proposed on the basis of one or more lines of evidence (9)(10)(11)13). These structural proposals have led to a number of mechanistic proposals for the role of calcium in O 2 evolution reviewed elsewhere (11,(14)(15)(16)(17).Dissection of the functional roles of the inorganic cofactors has come from in Vitro studies of the photoactivation process, which is defined as the assembly of the inorganic core during biogenesis and repair of the WOC. In Vitro photoactivation of the WOC is described by the following ...

Photoassembly of the water-oxidizing complex in photosystem II

“…Photoactivation kinetics and EPR spectroscopy reveal that when Ca 2+ binds in the dark to its specific site it induces the formation of ions, forming the active water oxidase. The photoactivation and spectroscopic data noted above provide a self-consistent picture locating the Ca effector site as an integral part of the Mn 4 core, in direct association with Mn probably via shared bridging ligands to Mn [28], [61].…”

“…Ca 2+ bound intermediates-We used EPR spectroscopy to characterize the ligand coordination environment of the first photoactivation intermediate (IM 1 and IM 1 * in Fig 5), corresponding to a photooxidized Mn 3+ species bound to apo-WOC-PSII [61]. The interconversion of the intermediates detected by EPR are schematically shown in Fig 4. In the absence of any added Ca 2+ , this species was shown to exist in two pH-dependent forms differing in terms of strength and symmetry of their ligand fields and whose populations are in equilibrium via a pH-dependent process.…”

Calcium controls the assembly of the photosynthetic water-oxidizing complex: a cadmium(II) inorganic mutant of the Mn 4 Ca core

“…Independent evidence in support of this model comes from studies of flash-induced pH changes showing that a proton is released at IM 1 Ã , either upon Ca 2C binding or Mn 2C photooxidation (Ananyev et al 2001). This same structural model for conversion of IM 1 /IM 1 Ã was inferred based upon EPR spectroscopic data describing the ligand field at Mn 3C and its environment (Tyryshkin et al 2006 (s) . The latter is measured by the large difference in their heats of formation DH f 0 ZK258 versus 635 kJ mol K1 , respectively (Frenkel 2005).…”

“…On the other hand, increasing the amount of Mn 2C by threefold to 30 mM did not produce any change in the cadmium inhibition profile. The latter concentration of Mn 2C is sufficient to appreciably change the fraction of PSII centres with bound Mn 2C , as it is nearly equal to the dissociation constant for Mn 2C at the high-affinity manganese site (K d Z0.45 mM; Tyryshkin et al 2006). The data in figures 1 and 2 indicate that Cd 2C binds to the calcium effector site required for photoactivation and not to the high-affinity Mn 2C site.…”

ESEEM Spectroscopy Reveals Carbonate and an N‐Donor Protein‐Ligand Binding to Mn2+ in the Photoassembly Reaction of the Mn4Ca Cluster in Photosystem II

“…[15] The EPR spectra in the presence of 13 C-and 12 C-bicarbonate are identical, indicating that the 13 C hyperfine couplings are small and therefore unresolved in the broad EPR linewidth. To spectrally resolve these small couplings, 2-pulse ESEEM experiments were carried out.…”