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. Author manuscript; available in PMC: 2020 Nov 6.
Published in final edited form as: J Am Chem Soc. 2019 Oct 28;141(44):17571–17587. doi: 10.1021/jacs.9b05715

Figure 6.

Figure 6.

ENDOR and three-pulse ESEEM spectra collected at 33.8 GHz on samples of W191Y Cpd I, W191Y:L232E Cpd I, and photogenerated tyrosyl solution. (A, B) Normalized 1H-ESE-ENDOR spectra of W191Y and W191Y:L232E Cpd I species and photogenerated tyrosyl in H2O and D2O 100 mM KPi buffer, pH 6; νLarmor ≈ 51 mHz. Subtracted spectra of Cpd I species (in dark and light gray) in protonated and deuterated environments reveal H-bonding interactions. Spectral interference with other weakly bound protons, however, makes quantification difficult. The asymmetric shoulder feature in the W191Y Cpd I difference spectrum is likely not significant. (C) Time domain spectra (vertically stacked and amplitudes normalized from 0 to 1 between 0 and 3000 ns) exhibit weaker stimulated-echo amplitude for W191Y Cpd I compared to similarly prepared samples of W191Y:L232E Cpd I and photogenerated Tyr. (D) Fourier-transformed signals show hyperfine couplings near that of the 2H Zeeman frequency. Signals normalized to the echo amplitudes at 0 ns demonstrate the lower levels of hydrogen bonding in W191Y Cpd I compared to W191Y:L232E.