Infrared crystallography of the Oxygen Evolving Complex reactions of Photosystem II

Understanding the molecular process of oxygen evolution is one of the great challenges in oxygenic photosynthesis research. Structural information is fundamental to aid and guide progress where recent breakthroughs have contributed substantially, particularly from X-ray crystallographic and EXAFS studies. However ambiguities exist in these recent structural studies in part because of X-ray radiation induced reduction and because the interpretation of polarised EXAFS measurements are not unique. Therefore a new structural methodology is needed in order to address these ambiguities and to provide added insight in the reactions at the pentanuclear Mn4Ca-cluster complex where water oxidation occurs and dioxygen is released. We propose novel measurements of Oxygen Evolving Complex (OEC) of PSII which overcome radiation problems and will give information not only about its structure but also its catalytic mechanism. Using linearly polarised infrared radiation focussed at the diffraction limit, the dichroic infrared absorption changes that accompany the S-state transitions in the OEC of single crystals of PSII isolated from Thermosynechococcus elongatus will be determined. This will yield vibrational transition dipole moment vectors of reactive groups in the X-ray frame and thus directly provides structural measurement of light induced changes of the Mn cluster and its ligands. This technique in effect amounts to infrared crystallography, which importantly does not cause radiation induced damage or reduction of high valency Mn in the OEC and as a structural technique will complement EXAFS and X-ray crystallography to probe the mechanistic reactions. There is a pressing need for such a novel structural technique to be applied to the important problem of photosynthetic water oxidation and the associated S-state transitions. Infrared crystallography of the OEC is a structural technique that has the added value to greatly enhance the analytical power of conventional unpolarised FTIR difference spectroscopy as well as complement X-ray diffraction and X-ray spectroscopy. The feasibility of such measurements have been demonstrated through preliminary experiments using existing PSII crystals. Additionally we have shown that the amplitudes of the signals from the active preparations will be significantly in excess of the detection limit of the proposed instrumentation. We propose the setting-up of a dedicated laboratory based instrument to exploit the technique and provide unique and important details of OEC functioning. Furthermore, developments in theoretical calculation of dipole gradients build on previous work in related areas and will support the interpretation of infrared dichroism in single crystals. The experimental availability within one experiment with internal consistency of frequencies, cross sections and dipole vectors in combination with rigorous theoretical modelling will provide structural and mechanistic insight into this important problem

We propose to construct and use for measurements a novel state-of-the-art instrument capable of reaction induced infrared difference dichroism measurements of very small crystals of Photosystem II that have suitable absorption characteristics. The goal is to obtain structural information on the OEC and spectroscopic information for further developing mode assignments, during S-state cycling and dioxygen production. Importantly, the infrared probe light has no reducing character in contrast to X-ray radiation used in X-rays crystallographic and EXAFS work, allowing full optical and thermal control of the reaction cycle of the OEC. The instrument will be based on a femtosecond Ti:Sapphire regenerative amplifier and TOPAS with DFM module for broadband infrared generation, an IR microscope and dispersive detection using LN2 cooled MCT arrays with full dispersive referencing and shot-to-shot normalisation. We will measure the dichroism of flash induced difference spectra of S-state transitions of suitable crystals of PSII and designed mutants with alterations in the OEC. Further, we shall perform modelling of molecular properties to support the interpretation of experimental observation of frequencies and dipole vectors of reactive groups in the OEC.