PULSED EPR STUDIES OF BIOLOGICAL MANGANESE CLUSTERS

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The molecular oxygen in our atmosphere is a product of a "water-splitting" reaction that occurs in the oxygen-evolving complex (OEC) of the Photosystem II component of plant photosynthesis. The catalytic core of the OEC is an ensemble of four manganese atoms arranged in a cluster of undetermined structure. Photosynthetic oxygen evolution occurs via a cycle that results in the removal of four electrons from two water molecules that bind to the Mn cluster. After this four-electron oxidation is complete, molecular oxygen is released, and the OEC returns to its initial state. This proposal concerns the use of novel pulsed electron paramagnetic resonance (EPR) methods designed to investigate this important enzyme system to a degree not possible with conventional continuous-wave EPR spectroscopy. The general goal is to utilize the pulsed EPR techniques of electron spin echo envelope modulation (ESEEM) and electron spin echo electron-nuclear double resonance (ESE-ENDOR) to measure the nuclear spin transitions of paramagnetic nuclei in protein ligands, cofactors, water molecules, and inhibitors bound to the Mn cluster, as well as spin transitions of the 55Mn nuclei of the cluster. The results will reveal new details concerning the structure and enzymatic function of this important biological metal center. More specifically, ESE-ENDOR experiments will be performed to measure 55Mn nuclear spin transitions of MN(III) and MN(IV) ions in a number of different synthetic exchange-coupled Mn clusters. Parallel experiments will be performed on the g=2 and g=4 Mn signals from the OEC in order to determine the degree to which each of the four Mn ions contributes to the EPR lineshapes. Other ESEEM and ESE-ENDOR experiments will measure spin transitions of the paramagnetic 17O isotope incorporated into oxygen bridges in the synthetic and biological clusters. Hyperfine and electric quadrupolar couplings will be determined for the 55Mn and 17O nuclei, and the results interpreted to provide new insights into the actual structure of the biological Mn complex. Corresponding experiments will be performed on the dinuclear Mn cluster of a manganese catalase enzyme. In addition, ESEEM and ESE-ENDOR experiments will be performed on Photosystem II membranes prepared with 1H20, 2H20, and H2 170-enriched buffers to determine at which stages of the oxygen-evolving cycle the two substrate water molecules bind to the cluster, and in what specific chemical form. ESEEM and ESE-ENDOR experiments will be performed to further characterize the protein ligation to the Mn cluster and to determine the proximity of the necessary cofactors Cl- and Ca2+ to the cluster. Additional pulsed EPR studies will determine if inhibitors of oxygen evolution that stabilize the g=4 Mn signal do so by binding directly to the Mn cluster.