The Mn 4 Ca Cluster in PSII DOI: 10.1002/ange.200702347 ESEEM Spectroscopy Reveals Carbonate and an N-Donor Protein- Ligand Binding to Mn 2+ in the Photoassembly Reaction of the Mn 4 Ca Cluster in Photosystem II** Jyotishman Dasgupta, Alexei M. Tyryshkin, and G. Charles Dismukes* The oxidation of water to dioxygen gas is an energy demanding and mechanistically complex chemical reaction. These limitations have restricted the development of practical water-splitting catalysts needed for renewable hydrogen production. By contrast in photosynthesis, water oxidation proceeds efficiently using visible light energy that is absorbed by chlorophyll photopigments and made chemically available through charge separation in the protein complex, photo- system II (PSII). [1] Water is oxidized and O 2 evolved ulti- mately at an inorganic active site (Mn 4 O x Ca 1 Cl y ) [2,3] known as the water oxidizing complex (WOC), by a mechanism that is debated. [4,5] X-ray structural evidence suggesting that inor- ganic carbon in the form of (bi)carbonate may serve as another required inorganic cofactor within the WOC has been circumstantial [2] and disputed. [6] However, studies have shown that (bi)carbonate unquestionably participates in the light- driven assembly of the inorganic core starting from the cofactor-depleted apo-WOC-PSII protein (called photoacti- vation). This is seen both by its acceleration of the rate of Mn 2+ photooxidation, [7,8] and by EPR spectroscopy of the resulting Mn 3+ assembly intermediate which revealed a strong influence on the strength of both the ligand field and the 55 Mn magnetic hyperfine coupling. [9] Although these observations indicate that (bi)carbonate acts to significantly alter the structural environment of the first Mn 3+ formed during photoassembly of the cluster and its electrochemical poten- tial, they have failed to identify where it actually binds within PSII. Thus, no direct evidence exists for the postulated inner coordination complex between bicarbonate and Mn 2+ during photoactivation. Herein, we provide evidence from electron spin echo envelope modulation (ESEEM) spectroscopy [10–12] for a direct magnetic hyperfine coupling between 13 C- bicarbonate and the Mn 2+ precursor to the photooxidized Mn 3+ formed in the first step of the photoactivation process. The photoactivation process occurs spontaneously upon illumination of the cofactor depleted apo-WOC-PSII protein in the presence of the free cofactors (Mn 2+ , Ca 2+ , Cl  ), visible light, and an electron acceptor. [13] Kinetic analysis of the assembly process has shown that it occurs in two resolved steps. The first intermediate is described by the binding of one Mn 2+ ion and its photo-oxidation to Mn 3+ . [14,23] Figure 1 shows the changes in the six-line absorption EPR lineshape of Mn 2+ in apo-WOC-PSII in the presence of 10 mm 12 C- and 13 C-bicarbonate upon illumination at 20 8C, recorded using field-sweep electron spin echo-detected EPR. This concentration of bicarbonate was chosen as it provides full saturation of the bicarbonate site determined from previous photoactivation studies and Mn 3+ EPR stud- ies. [7,16] In addition to the partially resolved six-line hyperfine pattern arising from Mn 2+ , the spectrum constitutes an overlapping radical signal at g = 2.0 arising from the photo- oxidized tyrosine radical Y D C . Upon illuminating the sample at 20 8C, the Mn 2+ intensity decreases in both the 12 C- and 13 C- bicarbonate samples to an equal extent (Figure 1). This change in the intensity corresponds to photooxidation of Figure 1. Field-sweep EPR spectra of Mn 2+ in apo-PSII under dark and after illumination at 20 8C, measured by using the 2-pulse ESE technique. In the presence of 10 mm NaH 12 CO 3 (solid black trace) and 10 mm NaH 13 CO 3 (dashed black line); upper and lower traces shown before (that is, in the dark) and after illumination. The 2-pulse ESEEM was collected at the field position indicated by the arrow. Conditions: interpulse delay t = 400 ns, pulse p/2 = 16 ns, temperature 5 K. [*] Dr. J. Dasgupta, Prof. G. C. Dismukes 7 Hoyt Laboratory Chemistry Department Princeton University Princeton, NJ 08544 (USA) Fax: (+ 1) 609-258-3449 E-mail: dismukes@princeton.edu Dr. A. M. Tyryshkin Department of Electrical Engineering Princeton University Princeton, NJ 08544 (USA) [**] We thank Dr. V.V. Klimov and Dr. Warwick Hillier for discussions. J.D. thanks the NASA Astrobiology Institute for financial support. This work was supported by grants from NIH GM-39932. ESEEM = electron spin echo envelope modulation. Supporting information for this article (time-domain 2-pulse ESEEM decays; 2-pulse ESEEM spectra measured at additional magnetic fields; 3-pulse ESEEM spectra of no added and 10 mm 13 C-bicarbonate added apo-WOC-PSII samples; and simulation parameters for 13 C-ESEEM spectrum) is available on the WWW under http://www.angewandte.org or from the author. Zuschriften 8174 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2007, 119, 8174 –8177