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

Abstract

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)-methanethiosulfo-nate] attached at position 81 of Cc (SLÀ Cc). A dissociation constant K D of 20 � 4 × 10 À 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.

Nitrogen-Vacancy Magnetic Relaxometry of Nanoclustered Cytochrome C Proteins

“…Numerous techniques have been utilized to detect Cyt-C, including electron paramagnetic resonance (EPR) spectrometry − and X-ray photoelectron spectroscopy (XPS). , The variation of the Cyt-C concentration in the cells depending on its current physiology state makes it hard to use bulk techniques such as EPR to detect it at the subcellular level. Among the currently available high-sensitivity and spatial resolution detection techniques, magnetic imaging based on the nitrogen-vacancy (NV) center in diamond − emerges as one of the most promising tools for high-resolution nanoscale imaging.…”