Effect of Mutantions and Labeling on Structural Stability of PpcA-Ru(bpy)3 Complexes

Abstract

Light-driven water oxidation in algae, cyanobacteria and higher plants generates dioxygen that supports life on Earth. The water-oxidation reaction is catalyzed by the oxygen-evolving complex (OEC) in photosystem II (PSII) that is comprised of the tetranuclear manganese calcium-oxo (Mn 4 Ca-oxo) cluster, redox-active tyrosine residue (Y Z ) and hydrogen-bonded network of amino acids and water molecules. The redox-active tyrosine residue, Y Z , mediates successive proton-coupled electron transfer (PCET) reactions that are essential for the oxidation of water to dioxygen at the Mn 4 Ca-oxo cluster. It is proposed that the strong hydrogen bond between Y Z and and its conjugate base, D1-His190, likely renders Y Z kinetically competent, leading to highly efficient water oxidation. However, a detailed understanding of PCET at Y Z remains elusive due to the lack of high-resolution structural methods to directly probe the electron-and proton-transfer reactions. In this study, we utilize highresolution two-dimensional (2D) 14 N and 1 H hyperfine sublevel correlation (HYSCORE) spectroscopy to investigate the electronic structure of the bioinspired artificial reaction center, benzimidazole-phenol porphyrin, that mimics PCET at the Y Z residue of PSII. Additionally, we perform density functional theory (DFT) calculations to determine the electron spin density distribution and electron-nuclear hyperfine coupling parameters of the benzimidazolephenol porphyrin radical for comparison with the corresponding parameters that are obtained from the 2D HYSCORE measurements. Redox-active proteins covalently modified with photosensitizers have served as important model systems for elucidation of kinetic principles controlling biological electron transfer. To date, the fastest rates observed in these systems were 10ns and involved donor acceptor (DA) distances of >0.8nm. This leaves a significant gap in rates and distances for charge transfer rates occurring in Nature but poorly accessible in experimental studies. To this end, we developed a series of 22 cysteine mutants of PpcA, a 3-heme cytochrome from Geobacter sulfurreducens. Due to its relatively small size (71 amino acids) we expected to obtain a number of mutants with DA distance of 0.4-0.5nm. These proteins were successfully expressed in E.coli and isolated for covalent labeling with Ru(bpy)2(bpy-Br). With time-resolved nanosecond and ultrafast transient absorbance spectroscopy we have identified 6 constructs with apparent photo-induced charge transfer time constants of 20 ps or faster, including 2 constructs with 1-2 ps time constants. The is a significant result as up to this point only natural photosynthetic systems demonstrated such a fast initial charge separation, while all artificial covalent biohybrid constructs exhibited charge transfer rates 3 or more orders of magnitude slower. To understand molecular principles responsible for such a dramatic acceleration of electron transfer rates, we used small-and wide angle X-ray scattering and currently attempting to obtain X-ra...