Ca²⁺ Depletion from the Photosynthetic Water-Oxidizing Complex Reveals Photooxidation of a Protein Residue

A new intermediate in the water-oxidizing reaction has been observed in spinach photosystem II (PSII) membranes that are depleted of Ca²⁺ from the site which is conformationally coupled to the manganese cluster comprising the water-oxidizing complex (WOC). It gives rise to a recently identified EPR signal (symmetric line shape with width 163 ± 5 G, g = 2.004 ± 0.005), which forms in samples inhibited either by depletion of Ca²⁺ [Boussac, A., Zimmerman, J.-L., & Rutherford, A. W. (1989) Biochemistry 28, 8984–8989; Sivaraja, M., Tso, J., & Dismukes, G. C. (1989) Biochemistry 28, 9459–9464] or by substitution of C1 by F⁻ (Baumgarten, Philo, and Dismukes, submitted for publication). Further characterization of this EPR signal has revealed the following: (1) it forms independently of the local structure of the PSII acceptors; (2) it arises from photooxidation of a PSII species that donates an electron to Tyr-Z⁺ or to the Mn cluster in competition with an exogenous donor (DPC); (3) the Curie temperature dependence of the intensity suggests an isolated doublet ground state, attributable to a spin S = ^↑/₂ radical; (4) the electron spin orientation relaxes 1000-fold more rapidly than typical for a free radical, exhibiting a strong temperature dependence of P_1/2 (half-saturation power ~T^3,4) and a broad inhomogeneous line width; (5) it yields an undetectable change in the magnetic susceptibility upon formation by a laser flash; (6) it disappears in parallel with release of Mn during reduction with NH₂OH, indicating that it forms only in the presence of the modified Mn cluster. One possible interpretation attributes this EPR signal to an unusual state of the Mn cluster not previously observed in synthetic Mn₂, Mn₃, or Mn₄ clusters. However, the most likely possibility is a radical which is in dipolar contact with a rapidly relaxing spin center such as a transition ion. We attribute this to photooxidation of an amino acid residue located within 9 Å of the Mn cluster. Starting in dark-adapted samples exhibiting the modified multiline EPR signal (apparent S₂′ oxidation state), the 160-G EPR signal forms with variable yield (24–48%) on the first flash and in >8O% centers after two saturating flashes. It thus appears associated with both the S₃′ and S₄′ states. This signal decays in the dark to the S₂′ state. The radical signal slowly disappears upon multiple turnovers during continuous illumination and may be correlated with the onset of photoinhibition-the irreversible loss of O₂ evolution. Photoinhibition occurs at a 17-fold faster rate than in normal PSII centers. We consider evidence suggesting that photooxidation of this species may occur during the normal mechanism of water oxidation.