Nanoscopic Understanding of Electron-Transfer and Channel-Forming Proteins in Action: A Multi-Frequency High-Field EPR Approach
Analytical Chemistry
Final Report Abstract
As part of a cooperation project of EPR groups in Berlin, Osnabrück/Dortmund, Mülheim a.d. Ruhr and Leiden (Holland) interdisciplinary research was pursued on high-field EPR spectroscopy on photosynthetic electron-transfer reaction center proteins and ion-channel forming protein toxins while passing through transient states of biological action. Geometrical and electronic structures of specific protein domains as well as their conformational changes and refolding dynamics were studied to understand biological function. Nanoscopic details of structure and lifetimes of the transient reaction intermediates, ranging from nanoseconds to seconds, were elucidated from the experimentally determined parameters and their quantum-chemical interpretation. Specifically, the research focused on the sub-projects: 1) Novel mutants of photosynthetic bacteria Rb. sphaeroides to analyze the mechanism of vectorial transmembrane electron transfer. 2) Site-directed spin-labeled mutants of the Colicin A bacterial toxin to analyze the refolding mechanism upon membrane insertion for ion-channel formation. 3) Instrumental improvements of the laboratory-built 95 and 360 GHz high-field EPR spectrometers to up-grade their sensitivity and time resolution to a level mandatory for this research. Taking sub-project 1) as example, it is fair to state that solar energy conversion by photosynthesis is the most important chemical process on Earth, from which almost all energy resources come and all the oxygen we breath. Essential ingredients of our present understanding of photosynthetic solar energy conversion have been discovered by spectroscopy performed at vastly different wavelengths. among them multi-frequency EPR spectroscopy. In photosynthetic organisms the light-induced electron- transfer processes across the membrane are uni-directional in nature. Presumably, subtle cofactor-protein interactions and/or conformational changes of specific protein segments are functionalized as molecular switches or electron gates. For an understanding of protein reactions on a nanoscopic level, the special and electronic structures of the initial, intermediate and final states are of primary concern. Distance and orientation of functional groups within the domains and their reaction-induced conformational changes determine the process efficiency, as does the fine-tuning of the reactants by weak interactions with the "solvent" matrix. EPR spectroscopy at high magnetic fields was applied to achieve the necessary sensitivity, resolution and orientational selectivity for the disordered protein samples. Ion radicals and radical pairs of the transient reaction intermediates of the pigment cofactors in reaction centers from wild-type and site-specific mutants of Rb. sphaeroides were studied by 95 GHz and 360 GHz EPR and double-resonance extensions thereof to characterize the cofactors' structure and dynamics in the hydrogen-bond network of their protein binding sites. The results are complementing the information of the 3D structure obtained from Xray protein crystallography on the stable ground-state conformation of the reaction center. The goal of such molecular-structure studies is a better understanding of the strategies Nature employs to optimize the light-induced primary charge separation steps in photosynthesis. The strategies are apparently based on a fine-tuning of the lifetimes of the transient intermediates to achieve longlived charge separation. Such an understanding on the molecular level is extremely important for developing optimization strategies in synthetic chemistry to mimic primary photosynthesis by artificial donor-acceptor complexes with efficient solar energy conversion. Sustaining success of such strategies would strongly contribute to the world-wide efforts to develop novel carbon-neutral renewable energy sources.
