Protein Scaffolds Modulate the Electronic Structure of a Common Ketyl Radical Intermediate in Thiamine Pyrophosphate-Dependent Radical Biocatalysis: Insights from EPR and ENDOR Spectroscopy

New-to-nature radical biocatalysis has emerged as a powerful strategy for asymmetric C–C bond formation, yet the enzyme-bound radical intermediates that underpin these transformations have remained largely uncharacterized. Here, we combine photobiocatalytic reaction development with multifrequency electron paramagnetic resonance (EPR), electron–nuclear double resonance (ENDOR) spectroscopy, and density functional theory (DFT) calculations to elucidate the ketyl radical intermediates formed during thiamine pyrophosphate (TPP)-dependent radical biocatalysis. We first develop an oxidative photobiocatalytic cross-coupling of aldehydes with alkyltrifluoroborates catalyzed by engineered benzoylformate decarboxylase (BFD) variants, providing enantioenriched α-chiral ketones through a new oxidative radical manifold inaccessible to benzaldehyde lyases (BALs). Freeze-quench multifrequency EPR and ENDOR spectroscopy directly identify the catalytically generated TPP-bound ketyl radical and reveal that this common radical intermediate adopts distinct electronic structures in BFD and BAL despite their identical chemical composition. Analysis of 1H, 19F, 31P, and 14N hyperfine interactions, together with DFT calculations, demonstrates that enzyme-specific active-site conformations redistribute spin density between the TPP cofactor and substrate fragment. Greater substrate-centered spin delocalization in BFD provides a mechanistic basis for its superior radical C–C coupling activity relative to BAL. These results establish a direct relationship between protein-controlled electronic structure and catalytic function in TPP-dependent radical biocatalysis and demonstrate that engineering the electronic properties of a common radical intermediate represents a useful strategy for controlling enzyme reactivity in radical biocatalysis.