HYSCORE Insights into the Distribution of the Unpaired Spin Density in an Engineered Cu _A Site in Azurin and Its His120Gly Variant

A comparative study of the ¹ H and ¹⁴ N hyperfine interactions between the Cu _A site in an engineered Cu _A center in azurin (WT-Cu _A Az) and its His120Gly variant (H120G-Cu _A Az) using the two-dimensional ESEEM technique, HYSCORE, is reported. HYSCORE spectroscopy has clarified conflicting results in previous electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies and found clear differences between the two Cu _A azurins. Specifically, a hyperfine coupling A _N of 15.3 MHz was determined for the first time from the frequencies of double-quantum transitions of ¹⁴ N histidine nitrogens coordinated to Cu _A in WT-Cu _A Az. In contrast, such coupling was not observed in the spectra of H120G-Cu _A Az, indicating at least a several megahertz increase in A _N for the coordinated nitrogen in this variant. In addition, ¹⁴ N HYSCORE spectra of WT-Cu _A Az show interaction with only one type of weakly coupled nitrogen assigned to the remote N _ϵ atom of coordinated imidazole residues based on the quadrupole coupling constant (e ² Qq/4h) of ∼0.4 MHz. The spectrum of H120G-Cu _A Az resolves additional features typical for backbone peptide nitrogens with larger e ² Qq/4h values of ∼0.7 MHz. Hyperfine couplings with these nitrogens vary between ∼0.4 and 0.7 MHz. In addition, the two resolved cross-peaks from C _β protons in H120G-Cu _A Az display only ∼1 MHz shifts relative to the corresponding peaks in WT-Cu _A Az. These new findings have provided the first experimental evidence of the previous density functional theory analysis that predicted changes in the delocalized electron spin population of ∼0.02-0.03 (i.e., ∼10%) on copper and sulfur atoms of the Cu _A center in H120 variants relative to WT-Cu _A Az and resolved contradicting results between EPR and ENDOR studies of the valence distribution in Cu _A Az and its variants.