Studies on 100% turnover higher plant photosystem II, as revealed by 55Mn EPR and Davies ENDOR

Abstract

Photosystem II or PS II, found in oxygenic photosynthetic organisms such as cynobateria or higher plants, is the catalyst for the most energetically demanding reaction in nature, the oxidation of water to molecular oxygen and protons. The water oxidase in PS II contains a Mn4Ca cluster (oxygen evolving complex, OEC), whose catalytic mechanism, despite extensive investigation, remains unresolved. The precise oxidation levels of the manganese is especially important in understanding the real catalytic mechanism of the OEC. Many experiemental techniques, such as EPR and ENDOR, have been adopted in historical studies, and also due to the more recent development in semiconductors, a higher level of computational analysis and simulation became available to study the system in theory. Here is the work completed to provide the first 55Mn pulsed ENDOR studies on the S2 state multiline spin ½ centre of the oxygen evolving complex (OEC) in Photosystem II (PS II), at temperatures below 4.2 K. These were performed on highly active samples of spinach PS II core complexes, developed previously in the laboratories using specific preparation procedure and experimental techniques to achieve 100% turnover rate, for photosystem spectroscopic use, at temperatures down to 2.5 K. Under these conditions, previously hindered observation, by relaxation effects, of most of the manganese ENDOR resonances from the OEC coupled Mn cluster are suppressed.55Mn ENDOR hyperfine couplings ranging from 50 to 680 MHz are now seen on the S2 state multiline EPR signal. These, together with complementary high resolution X-band CW EPR measurements and detailed simulations, reveal that at least two and probably three Mn hyperfine couplings with large anisotropy are seen, indicating that three MnIII ions are likely present in the functional S2 state of the enzyme. This suggests a low oxidation state paradigm for the OEC (mean Mn oxidation level 3.0 in the S1 state) and unexpected Mn exchange coupling in the S2 state, with two Mn ions nearly magnetically silent. Our results rationalize a number of previous ligand ESEEM/ENDOR studies and labelled water exchange experiments on the S2 state of the photosystem.