Direct detection of oxygen ligation to the Mn(4)Ca cluster of photosystem II by X-ray emission spectroscopy.

Direct Detection of Oxygen Ligation to the Mn 4 Ca Cluster of Photosystem II by X-ray Emission Spectroscopy Yulia Pushkar †,# , Xi Long †,‡ , Pieter Glatzel †,€ , Gary W. Brudvig § , G. Charles Dismukes ¶ , Terrence J. Collins $ , Vittal K. Yachandra †,* , Junko Yano †,* , Uwe Bergmann ∆,* Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, § Dept. of Chemistry, Yale Univ., New Haven, CT, ¶ Dept. of Chemistry, Princeton Univ., Princeton, NJ, $ Dept. of Chemistry, Carnegie-Mellon Univ., Pittsburgh, PA, ∆ Stanford Synchrotron Radiation Lightsource, Menlo Park, CA. RECEIVED DATE (automatically inserted by publisher); vkyachandra@lbl.gov, jyano@lbl.gov, ubergmann@slac.stanford.edu Ligands play critical roles during the catalytic reactions in metalloproteins through bond formation/breaking, protonation/deprotonation, and electron/spin delocalization. While there are well-defined element-specific spectroscopic handles, such as X-ray spectroscopy and EPR, to follow the chemistry of metal catalytic sites in a large protein matrix, directly probing particular ligand atoms like C, N, and O is challenging due to their abundance in the protein. FTIR/Raman and ligand-sensitive EPR techniques such as ENDOR and ESEEM have been applied to study metal-ligand interactions. X-ray absorption spectroscopy (XAS) can also indirectly probe the ligand environment; its element-specificity allows us to focus only on the catalytic metal site, and EXAFS and XANES provide metal-ligand distances, coordination numbers, and symmetry of ligand environments. However, the information is limited, since one cannot distinguish among ligand elements with similar atomic number (i.e. C, N. and O). As an alternative and a more direct method to probe the specific metal-ligand chemistry in the protein matrix, we investigated the application of X-ray emission spectroscopy (XES). Using this technique we have identified the oxo-bridging ligands of the Mn 4 Ca complex of photosystem II (PS II), a multisubunit membrane protein, that catalyzes the water oxidizing reaction. 1 The catalytic mechanism has been studied intensively by Mn XAS. 2 The fundamental question of this reaction, however, is how the water molecules are ligated to the Mn 4 Ca cluster and how the O-O bond formation occurs before the evolution of O 2 . 3-5 This implies that it is necessary to follow the chemistry of the oxygen ligands in order to understand the mechanism. XES which is a complementary method to XAS, has the potential to directly probe ligation modes. 6 Among the several emission lines, Kβ 1,3 and Kβ′ lines originate from the metal 3p to 1s transition, and they have been used as an indicator of the charge and spin states on Mn in the OEC (Figure 1). 7,8 The higher energy region corresponds to valence to core transitions just below the Fermi level, and can be divided into the Kβ′′ and the Kβ 2,5 emission (Fig.1 left scheme). Kβ 2,5 emission is predominantly from ligand 2p (metal 4p) to metal 1s, and the Kβ′′ emission is assigned to a ligand 2s to metal 1s, and are referred to as crossover transitions. 9-11 Therefore, only direct ligands to the metal of interest are probed with Kβ ,2,5 /Kβ′′ emission; i.e. other C, N, and O atoms in the protein media do not contribute to the spectra. In this report, we focus on the Kβ′′ spectral region to characterize metal-ligand interactions, in particular contributions from ligated oxygens. The energy of the Kβ′′ transition is dependent on the difference between the metal 1s and ligand 2s binding energies, which is dependent on the environment of the Present addresses: # Dept. of Physics, Purdue Univ., West Lafayette, IN 47904; ‡ Dept. of Chemistry, Univ. of California, Santa Cruz, CA 95064; ESRF, BP 220, 38043 Grenoble Cedex, France. Figure 1. (A) Energy diagram of Mn Kβ transitions in MnO. The Kβ′′ and Kβ 2,5 transitions are from valence molecular orbitals, Kβ′′ is O 2s to Mn 1s ‘cross-over’ transition. (B) Logarithmic plot of MnO Kβ spectrum. The O-Mn cross-over Kβ′′ transition is highlighted. ligand due to orbital hybridization. Therefore the Kβ′′ energy is affected by the charge density on the metal, the ligand protonation state, and changes in the coordination environment. The Kβ′′ intensity is influenced by the spatial overlap between the wavefunction that describes the Mn 1s orbital and the molecular orbitals on the ligands. The Kβ′′ intensity is affected by the metal to ligand distance, and the number of ligands per metal ion. Shorter distances (e.g. from higher bond order or deprotonation) result in increased Kβ′′ intensity with an approximate exponential dependence. 9 On the other hand, a spread of the molecular wavefunction over next-nearest neighbor atoms will decrease the Kβ′′ spectral intensity. Therefore contribution from single atom ligands such as oxo-bridges, or terminal oxo ligands bonded to Mn is predominant (see below). These combination of factors makes the Kβ′′ spectrum a powerful tool for detection and characterization of oxo-bridges in the Mn 4 Ca cluster of PS II. However, because of the weak intensity of the Kβ′′ spectrum obtaining such spectra from biological samples as dilute as PS II (800µM Mn) has been difficult. For O ligation in a typical model compound, the signal is ~10 3 times weaker than that of Kα and there is an additional large background from both the Kβ 1,3 and the Kβ 2,5 spectral features (Fig. 1). Furthermore the work is challenging because of the high sensitivity of the Mn 4 Ca cluster to radiation damage. 12 This study of PS II became possible by using a new high resolution spectrometer equipped with 8-14 analyzer crystals collecting a large solid angle (Suppl. Info.). Fig. 2 shows the Kβ′′ spectrum of a sample of PS II in the S 1 state compared with a series of Mn oxide spectra. Each spectrum is normalized by the Kβ 1,3 peak intensity which is proportional to the number of Mn atoms in the system. The 1 st moment energy of