The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy

We present a novel approach to study transient protein‐protein complexes with standard, 9 GHz, and high‐field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso‐1‐cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1‐oxyl‐2,2,5,5‐tetramethyl‐Δ3‐pyrroline‐3‐methyl)‐methanethiosulfonate] attached at position 81 of Cc (SL−Cc). A dissociation constant K(D) of 20±4×10(−6) M (EPR and NMR) and an equal amount of stereo‐specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc‐interaction surface, with the stereo‐specific complex.