Determination of the Complete Spin Density Distribution in C-Labeled Protein-Bound Radical Intermediates Using Advanced 2D Electron Paramagnetic Resonance Spectroscopy and Density Functional Theory.

Determining the complete electron spin density distribution for protein-bound radicals, even with advanced pulsed electron paramagnetic resonance (EPR) methods, is a formidable task. Here we present a strategy to overcome this problem combining multifrequency HYSCORE and ENDOR measurements on site-specifically C-labeled samples with DFT calculations on model systems. As a demonstration of this approach, pulsed EPR experiments are performed on the primary Q and secondary Q ubisemiquinones of the photosynthetic reaction center from Rhodobacter sphaeroides C-labeled at the ring and tail positions. Despite the large number of nuclei interacting with the unpaired electron in these samples, two-dimensional X- and Q-band HYSCORE and orientation selective Q-band ENDOR resolve and allow for a characterization of the eight expected C resonances from significantly different hyperfine tensors for both semiquinones. From these results we construct, for the first time, the most complete experimentally determined maps of the s- and p-orbital spin density distributions for any protein organic cofactor radical to date. This work lays a foundation for understanding the relationship between the electronic structure of semiquinones and their functional properties, and introduces new techniques for mapping out the spin density distribution that are readily applicable to other systems.