Study of human Cytochrome P450 reductase by Electron Paramagnetic Resonance

A novel doubly rotating frame (DRF) technique has been exploited to calculate three- and four-pulse DEER signals for a system of two dipolar-coupled nitroxides. The three-pulse DRF technique is illustrated for the case, wherein the relative intensity of the peak, appearing at ±d/2, is compared with that of the peak at ±d, where d=23D (D = dipolar-coupling constant, as a function of B1, the amplitude of the pulses. It is found for three-pulse DEER signals that for the commonly used B1 fields the three-pulse DEER sequence is capable of measuring distances between the two nitroxides of the biradical corresponding to d≤8 MHz, or distances r≥ 18.7 Å. As for the four-pulse DEER signal, using the distribution of the distance between the nitroxide radicals, determined experimentally, the simulated DEER signal shows excellent agreement with the experiment.