Solution NMR study of the yeast cytochrome c peroxidase: cytochrome c interaction
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Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc–CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.
KeywordsTransient complex Macromolecular recognition Binding shifts Electron transfer
We thank Karen Van de Water for help with analysis of the low-salt data and Yann Sterckx and Sophie Vanwetswinkel for the critical reading of the manuscript. A. N. V. is an FWO Post-Doctoral Researcher. We acknowledge the financial support from VIB and the Hercules Foundation.
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