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.
Bibliographical noteFunding Information:
This investigation was supported in part by Grant DE-FG02-08ER15960 (S.A.D., pulsed EPR work) from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, U.S. Department of Energy, and National Science Foundation Grant CHE 17-10241 (Y.L., protein design and engineering). The authors thank Dr. Alexander Taguchi (Massachusetts Institute of Technology, Cambridge, MA) for his help with the simulation of HYSCORE spectra.
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