Cytochrome c in a dry trehalose matrix: structural and dynamical effects probed by X-ray absorption spectroscopy

¹ Dipartimento di Fisica, Università di Bologna
² Dipartimento di Biologia, Università di Bologna
³ Dipartimento di Scienze Fisiche ed Astronomiche, Università di Palermo

^* To whom correspondence should be addressed. E-mail: ventur{at}alma.unibo.it.

Submitted on June 27, 2006
Revised on July 27, 2006
Accepted on 1 November 2006

	Abstract

We report on the structure and dynamics of the Fe ligand cluster of reduced horse heart cytochrome c in solution, in a dried polyvinyl alcohol (PVA) film and in two trehalose matrices characterized by a different content of residual water. The effect of the solvent/matrix environment was studied at room temperature using Fe K-edge X-ray absorption fine structure (XAFS) spectroscopy. XAFS data were analysed by combining ab-initio simulations and multiparameter fitting, attempting to disentangle structural from disorder parameters. Essentially the same structural and disorder parameters account adequately for the XAFS spectra measured in solution, both in the absence and in the presence of glycerol, and in the PVA film, showing that this polymer interacts weakly with the embedded protein. Instead, incorporation in trehalose leads to severe structural changes, more prominent in the more dried matrix, consisting in: (a) an increase up to 0.2 Å of the distance between Fe and the imidazole N atom of the coordinating histidine residue; (b) an elongation up to 0.16 Å of the distance between Fe and the forth shell C atoms of the heme pyrrolic units. These structural distortions are accompanied by a substantial decrease of the relative mean square displacements of the first ligands. In the extensively dried trehalose matrix extremely low values of the Debye Waller factors are obtained for the pyrrolic and for the imidazole N atoms. This finding is interpreted as reflecting a drastic hindering in the relative motions of the Fe ligand cluster atoms and an impressive decrease in the static disorder of the local Fe structure. It appears therefore that the dried trehalose matrix perturbs dramatically the energy landscape of cyrochrome c, giving rise, at the level of local structure, to well resolved structural distortions and restricting the ensemble of accesible conformational substates.

Key Words: XAFS, heme proteins, protein dynamics, protein-solvent interactions, trehalose

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