Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidase determined by paramagnetic NMR

doi:10.1073/pnas.0603551103

Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidase determined by paramagnetic NMR

Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands

Edited by Brian M. Hoffman, Northwestern University, Evanston, IL, and approved October 18, 2006 (received for review May 1, 2006)

Abstract

The physiological complex of yeast cytochrome c peroxidase and iso-1-cytochrome c is a paradigm for biological electron transfer. Using paramagnetic NMR spectroscopy, we have determined the conformation of the protein complex in solution, which is shown to be very similar to that observed in the crystal structure [Pelletier H, Kraut J (1992) Science 258:1748–1755]. Our results support the view that this transient electron transfer complex is dynamic. The solution structure represents the dominant protein–protein orientation, which, according to our estimates, is occupied for >70% of the lifetime of the complex, with the rest of the time spent in the dynamic encounter state. Based on the observed paramagnetic effects, we have delineated the conformational space sampled by the protein molecules during the dynamic part of the interaction, providing experimental support for the theoretical predictions of the classical Brownian dynamics study [Northrup SH, Boles JO, Reynolds JCL (1988) Science 241:67–70]. Our findings corroborate the dynamic behavior of this complex and offer an insight into the mechanism of the protein complex formation in solution.

Footnotes

^‡To whom correspondence should be addressed. E-mail: m.ubbink{at}chem.leidenuniv.nl
Author contributions: A.N.V., J.A.R.W., and M.U. designed research; A.N.V., J.A.R.W., and E.H. performed research; A.N.V., J.A.R.W., and M.U. analyzed data; and A.N.V. and M.U. wrote the paper.
↵*Present address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Old Addenbrookes Site, Cambridge CB2 1GA, United Kingdom.
↵ ^†Present address: Faculty of Pharmacy, Université Paris 5, 4 Avenue de l'Observatoire, 75006 Paris, France.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2GB8).
This article contains supporting information online at www.pnas.org/cgi/content/full/0603551103/DC1.
Abbreviations:

ET,

electron transfer;

Cc,

yeast iso-1-cytochrome c;

CcP,

yeast cytochrome c peroxidase;

PRE,

paramagnetic relaxation enhancement;

MTSL,

(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-methanethiosulfonate;

MTS,

(1-acetyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-methanethiosulfonate;

HSQC,

heteronuclear single-quantum coherence