Pulse field-sweep EPR of copper proteins

Pulse field-sweep EPR (PFSEPR) is developed as a low-power, microwave pulse technique to resolve hyperfine structure underlying inhomogeneously broadened EPR lines. PFSEPR arises from transfer of saturation due to spin state mixing from forbidden Δm_I ≠ 0 EPR transitions. We report its first use to resolve large copper hyperfine couplings which are unresolved by standard X-band EPR. As applied to copper, PFSEPR has better sensitivity than ENDOR with comparable spectral resolution. The details of energy levels and state mixing which account for PFSEPR transitions in these copper systems are developed here. PFSEPR transitions from stellacyanin provide hyperfine and quadrupole couplings in good agreement with those predicted by various EPR simulations and determined by ENDOR. Copper couplings from the Cu_A signal of cytochrome-c oxidase are comparable to previously published estimates, but PFSEPR suggests underlying state mixing of copper levels.