Accumulation and Pulse Electron Paramagnetic Resonance Spectroscopic Investigation of the 4-Oxidobenzyl Radical Generated in the Radical S -Adenosyl-l-methionine Enzyme HydG.

The radical S -adenosyl-l-methionine (SAM) enzyme HydG cleaves tyrosine to generate CO and CN ^- ligands of the [FeFe] hydrogenase H-cluster, accompanied by the formation of a 4-oxidobenzyl radical (4-OB ^• ), which is the precursor to the HydG p -cresol byproduct. Native HydG only generates a small amount of 4-OB ^• , limiting detailed electron paramagnetic resonance (EPR) spectral characterization beyond our initial EPR lineshape study employing various tyrosine isotopologues. Here, we show that the concentration of trapped 4-OB ^• is significantly increased in reactions using HydG variants, in which the "dangler Fe" to which CO and CN ^- bind is missing or substituted by a redox-inert Zn ²⁺ ion. This allows for the detailed characterization of 4-OB ^• using high-field EPR and electron nuclear double resonance spectroscopy to extract its g -values and ¹ H/ ¹³ C hyperfine couplings. These results are compared to density functional theory-predicted values of several 4-OB ^• models with different sizes and protonation states, with a best fit to the deprotonated radical anion configuration of 4-OB ^• . Overall, our results depict a clearer electronic structure of the transient 4-OB ^• radical and provide new insights into the radical SAM chemistry of HydG.