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

Abstract

We are developing biomimetic molecular architectures for efficient solar energy conversion using artificial photosensitizers combined with natural and genetically engineered host systems capable to support long-lived charge-separated states and conduct charges away from the photosensitizers. Converting light energy into its electrochemical equivalent requires precise control and fine tuning of relevant kinetic and thermodynamic parameters, including primary charge separation. To this end, we developed a series of 22 cysteine mutants of PpcA, a 3-heme cytochrome from Geobacter sulfurreducens. Protein mutants were labeled at the engineered Cys sites with Ru(bpy)3Br. We verified correct photosensitizer attachment and the extent of labeling with Liquid chromatography- mass spectroscopy (LC-MS). The correctly labeled complexes were analyzed with Circular Dichroism (CD) spectroscopy at various temperature points between 25 and 90°C. Contrary to the expectation that mutations and binding positively charged bulky photosensitizers will cause destabilization of protein structure, we observed relatively small perturbations for the majority of the mutants as well as two complexes more stable than the wild-type protein. Our results also reveal strong preference of individual mutants for binding Ru(bpy)3 enantiomers from racemic mixture. The obtained results are discussed in context of prior extensive Small-angle X-ray Scattering (SAXS) characterization and all-atom Molecular Dynamics (MD) simulations in explicit solvent.