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

A comparative study of the 1H and 14N hyperfine interactions between the CuA site in an engineered CuA center in azurin (WT-CuAAz) and its His120Gly variant (H120G-CuAAz) 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 CuA azurins. Specifically, a hyperfine coupling AN⊥ of 15.3 MHz was determined for the first time from the frequencies of double-quantum transitions of 14N histidine nitrogens coordinated to CuA in WT-CuAAz. In contrast, such coupling was not observed in the spectra of H120G-CuAAz, indicating at least a several megahertz increase in AN⊥ for the coordinated nitrogen in this variant. In addition, 14N HYSCORE spectra of WT-CuAAz show interaction with only one type of weakly coupled nitrogen assigned to the remote Nε atom of coordinated imidazole residues based on the quadrupole coupling constant ( e2 Qq/4 h) of ∼0.4 MHz. The spectrum of H120G-CuAAz resolves additional features typical for backbone peptide nitrogens with larger e2 Qq/4 h 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 Cβ protons in H120G-CuAAz display only ∼1 MHz shifts relative to the corresponding peaks in WT-CuAAz. 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 CuA center in H120 variants relative to WT-CuAAz and resolved contradicting results between EPR and ENDOR studies of the valence distribution in CuAAz and its variants.