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

• Published in: Chemphyschem Vol. 21; no. 10; pp. 1060 - 1069
• Main Authors: Son, Martin, Schilder, Jesika T., Di Savino, Antonella, Blok, Anneloes, Ubbink, Marcellus, Huber, Martina
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 18.05.2020; John Wiley and Sons Inc
• Subjects: Algorithms; Cytochrome; Cytochrome-c Peroxidase - chemistry; Cytochrome-c Peroxidase - metabolism; Cytochromes; Cytochromes c - chemistry; Cytochromes c - metabolism; Electron paramagnetic resonance; Electron Spin Resonance Spectroscopy; high-field EPR; Models, Molecular; NMR spectroscopy; Nuclear Magnetic Resonance, Biomolecular; paramagnetic NMR; Peroxidase; principal component analysis; Protein Binding; Proteins; transient protein complex; cytochrome; paramagnetic NMR; high-field EPR; transient protein complex; principal component analysis
• ISSN: 1439-4235; 1439-7641; 1439-7641
• DOI: 10.1002/cphc.201901160

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 KD 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. Transient protein‐protein complexes fleetingly encounter and efficiently react. They leave subtle traces in the shape of electron paramagnetic resonance (EPR) spectra, which – systematically analyzed – give nanosecond glimpses of the interaction of the two proteins. When paramagnetic NMR and EPR are combined, a picture emerges in which one can see the two proteins (blue and green shapes) not only in the stereo‐specific complex (dark green) but also in the encounter complex (light green). The spin label is shown in red.