Comparison of PELDOR and RIDME for Distance Measurements between Nitroxides and Low-Spin Fe(III) Ions

EPR-based nanometre distance measurements are becoming ever more important in structural biology. Usually the distance constraints are measured between two nitroxide spin labels. Yet, distance measurements between a metal center and spin labels enable, e.g., the localization of metal ions within the tertiary fold of biomolecules. Therefore, it is important to find methods that provide such distance information quickly, with high precision and reliability. In the present study, two methods, pulsed electron-electron double resonance (PELDOR) and relaxation-induced dipolar modulation enhancement (RIDME), are compared on the heme-containing and spin-labeled cytochrome P450cam. Special emphasis is put on the optimization of the dead-time free RIDME experiment and several ways of data analysis. It turned out that RIDME appears to be better suited for distance measurements involving metal ions like low-spin Fe3+ than PELDOR.