Pulsed Q-band two pulse EPR and 14N, 1H Davies ENDOR of PmoD. (A) Two pulse echo-detected EPR. The Cu2+ region denoted by the dotted line, g ~ 2.2-2.15, is attributable to exclusively mononuclear Cu2+ resonance, while the region from g ~ 2.15 – 2.0 corresponds to predominantly CuA resonance (as well as the overlapping minor Cu2+ resonance). Field-swept spectrum with X-axis of magnetic field provided in Figure S8. (B) 14N Davies ENDOR measurements across the EPR envelope at g-values indicated, demonstrating the nearly equivalent hyperfine coupling of the two CuA
14N ligands. The region under the dotted brackets denotes resonance attributable to Cu2+−14N ligation, as confirmed in Figure S5A. The black goalpost width signifies twice the 14N Larmor frequency (2 x v14N, and the filled circle denotes one half the 14N hyperfine coupling (A/2). Additional splitting resolved at the high field edge (g = 2.02) of the 14N2 resonance is attributed to resolved quadrupole splitting 3P = 2.3 MHz. Only the higher frequency v+ peaks are well-resolved. (C) 1H Larmor-centered Davies ENDOR, where the triangle denotes the 1H Larmor frequency (v1H) and goalpost width defines the hyperfine coupling magnitude (A) to CuA Cys-Cβ
1H (black). The modestly large 1H response seen at lower fields (A ~ 8 MHz) is attributed to a 1H coupled to the underlying Cu2+ resonance, as confirmed in Figure S5B. EPR conditions: 34.649 GHz microwave frequency, 200 s scan, π = 80 ns, τ = 500 ns, 20 ms repetition time; 14N ENDOR conditions: 34.63-34.67 GHz microwave frequency, π = 80 ns, τ = 375 ns, TRF = 200 μs, 20 ms repetition time; 1H ENDOR conditions: 34.64-34.65 GHz microwave frequency, π = 200 ns, τ = 575 ns, TRF = 60 μs, 50 ms repetition time.