Protein and Substrate Coordination to the Manganese Cluster in the Photosynthetic Water Oxidizing Complex: ¹⁵N and ¹H ENDOR Spectroscopy of the S₂ State Multiline Signal in the Thermophilic Cyanobacterium Synechococcus elongatus

The hyperfine constants have been measured by ENDOR spectroscopy for ¹H and ¹⁵N nuclei located within magnetic contact to the tetranuclear manganese cluster of the photosynthetic water oxidizing site in the thermophilic cyanobacterium Synechococcus el. The Mn cluster was examined in the S₂ oxidation state using the “multiline” EPR signal. The data were compared to model dimanganese(III,IV) complexes possessing both N and O ligand atoms and µ-oxo and µ-carboxylato bridging ligands. This revealed that the photosynthetic Mn cluster is coordinated predominantly by nonmagnetic O atoms having no covalently bound protons at α or β positions. This is indirect evidence for protein-derived carboxylato type ligands. Two ¹⁵N hyperfine constants were resolved at 0.7 and 3.7 MHz. These values are comparable to the range predicted for coordination to π type sites on Mn^III or Mn^IV and exclude σ type coordination sites on Mn^III which yield much larger hyperfine constants. Either a single class of protein N (imidazole) ligands with coupling to both N1 and N3 atoms or possibly two coordination sites could be involved. An unexpectedly simple ¹H ENDOR spectrum was observed with two well resolved hyperfine splittings of 2.4 and 1.0 MHz and two poorly resolved or weak splittings of 4.9 and 0.5 MHz. All of these were removed by incubation in ²H₂O. Positive assignment to the Mn cluster was established by ENDOR-induced EPR. The ¹H ENDOR results differ greatly from those reported for the Mn cluster in spinach (Kawamori, Inui, Ono, Inoue FEBS Lett. 1989, 254, 219–224). The three largest splittings could be accounted for by a simple model involving a single rhombic ¹H tensor, with four possible sign choices for the principal values. One of these choices, A_x,y,z(dipolar) = −4.5, 2.9, 1.5 and A(isotropic) = −0.5 (MHz), coincided with the predicted dipolar and isotropic hyperfine terms obtained from a spin-coupled point-pair model used to describe the ligand dipolar hyperfine interaction with a pair of spin-coupled paramagnetic ions. The experimental ENDOR line shape could be approximately simulated by location of a proton nearly equidistant between the Mn ions along the normal to the Mn-Mn vector (R). The closest approach is predicted for the case of antiferromagnetically coupled Mn^III and Mn^IV ions, for which the proton would be located at R = 3.65 Å. At the present level of sensitivity, the hyperfine data suggest there may be no un-ionized water or hydroxo ligands directly bound to the Mn cluster but instead a “dry” environment with rather long (weak) H-bonds from solvent exchangeable protons to µ-oxo bridges or possibly to terminally coordinated ligands.