Primary Processes in Photosynthesis: What do we learn from High-Field EPR Spectroscopy?

Abstract

Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.8 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), two classes of photosynthetic protein systems are characterized with respect to structure and dynamics: (i) Light-generated electron-transfer intermediates in reaction center proteins, for example from the Rb. sphaeroides purple bacterium, (ii) light-driven proton-pump intermediates of site-directed nitroxide spin-labeled bacteriorhodopsin proteins from the purple membrane of Halobacterium salinarium. The aim of theses studies is to obtain detailed molecular information beyond the X-ray structure for a better understanding of the structure-dynamics-function relationships of transfer proteins which convert energy of sunlight into electrochemical energy. (i) Primary photosynthesis in the reaction centers (RCs) of green plants and purple bacteria is the process of light-induced charge separation and stabilization of (bacterio) chlorophyll donor (P) and quinone acceptor (Q) cofactors via transmembrane electron-transfer steps. Thereby transient radical ions P+• and QA -• together with weakly coupled radical-pair states P+•Q-• are formed. For the P+•, QA -•and QB -• cofactors in their binding sites of Rb. sphaeroides RCs, cw and pulsed high-field EPR/ENDOR and field-swept electron-spinecho experiments provided detailed information on structure, hydrogen-bond interactions and anisotropic dynamics at biologically relevant time scales. (ii) The combination of EPR and genetic methods for selected mutations is a powerful strategy for determining structure and dynamics of proteins by site-directed spin labeling (SDSL) with one or two appropriately functionalized nitroxide radicals. For the light-induced proton-transfer protein bacteriorhodopsin the SDSL/EPR method at high Zeeman fields and microwave frequencies becomes particularly powerful for elucidating polarity and proticity effects of the protein microenvironment on hyperfine and g-tensors of nitroxide spin labels along putative proton pathways across the membrane. Conformational changes of the protein during the photocycle could be traced revealing location and function of the molecular switch for vectorial proton transfer. A short description of the laboratory-built 95-GHz and 360-GHz EPR and ENDOR spectrometers at FU Berlin is also included in this Chapter.Key wordsprimary photosynthesisplant and bacterial reaction centersproton pump bacteriorhodopsinsite-directed spin-labelinglight-induced conformational changeshigh-field EPR on proteins