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Cytochrome c in a Dry Trehalose Matrix: Structural and Dynamical Effects Probed by X-Ray Absorption Spectroscopy

Lisa Giachini ^* , Francesco Francia , Lorenzo Cordone  , Federico Boscherini ^*  and Giovanni Venturoli  

^* Dipartimento di Fisica, Università di Bologna, 40126 Bologna, Italy; Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), Italy; Laboratorio di Biochimica e Biofisica, Dipartimento di Biologia, Università di Bologna, 40126 Bologna, Italy; and Dipartimento di Scienze Fisiche ed Astronomiche, Università di Palermo, 90123 Palermo, Italy

Correspondence: Address reprint requests to Giovanni Venturoli, Laboratorio di Biochimica e Biofisica, Dipartimento di Biologia, Università di Bologna, 40126 Bologna, Italy. E-mail: ventur{at}alma.unibo.it.

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 different contents 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 analyzed by combining ab initio simulations and multi-parameter fitting in an attempt 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 of 1), an increase up to 0.2 Å of the distance between Fe and the imidazole N atom of the coordinating histidine residue and 2), an elongation up to 0.16 Å of the distance between Fe and the fourth-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 dramatically perturbs the energy landscape of cyrochrome c, giving rise, at the level of local structure, to well-resolved structural distortions and restricting the ensemble of accessible conformational substates.

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