1H HYSCORE Studies of Second Sphere Variants of the Type 1 Copper Site in Azurin: Influence of Mutations on the Hyperfine Couplings of Cys112 CβH2 Protons

In this work, we examined the influence of F114P, F114N, and N47S mutations on the electronic structure of the T1 copper center in azurin (Az) by studying the hyperfine couplings of C_βH₂ protons of Cys112 using the two-dimensional (2D) pulsed EPR technique HYSCORE (hyperfine sublevel correlation). Our results show that isotropic hyperfine couplings for the two Cys112 C_β protons in WTAz, F114NAz, and N47SAz vary within 21 ± 2 MHz, with differences of only several tenths of MHz between the two protons in a given protein despite major variations previously observed in S_C112 electron density. Furthermore, significantly smaller couplings of 0.7 MHz and 7.4 MHz are observed for the F114PAz mutation—which eliminates a backbone amide H-bonding interaction with S_C112—despite previous observations of a significant increase in S character in the SOMO, demonstrating a more complex relationship between C_β–¹H hyperfine and the unpaired spin density of S_C112 than previously proposed. In addition, the 2D HYSCORE spectra show resolvable differences in the anisotropic couplings between the two protons in all samples, indicating inconsistencies between previously reported ENDOR and computational data for the WTAz protein. Other findings visible in 2D spectra is the distortion of the cross-ridges from Cys112 C_βH₂ protons with the large isotropic couplings resulted from a_iso strain produced by the fluctuations of the C_β‒H₂ group orientation within θ ~  ± 2° degrees around the most populated conformation.