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

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 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 A of the distance between Fe and the imidazole N atom of the coordinating histidine residue and 2), an elongation up to 0.16 A 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 cytochrome c, giving rise, at the level of local structure, to well-resolved structural distortions and restricting the ensemble of accessible conformational substates.

FIGURE 1

(A) Reference structural model of the Fe ligand cluster (38). (B) Sketch of the structural units of the reference model. The values of distances and angle are the following: Heme pyrrolic unit: Fe-N_p = 1.99 Å; N_p-C_2,5 = 1.38 Å; C_2,5-C_3,4 = 1.44 Å; C₃-C₄ = 1.34 Å; C₂-C₆ = 1.38 Å; FeN_pC_2,5 = 109°; FeN_pC_3,4 = 163°; N_pC₂C₆ = 125°; Histidine imidazole: Fe-N_ɛ2 = 2.00 Å; N_ɛ2-C_ɛ1= 1.32 Å; N_ɛ2-C_δ2= 1.37 Å; C_ɛ1-N_δ1 = 1.34 Å; C_δ2-C_γ = 1.35 Å; C_γ-N_δ1 = 1.35 Å; FeN_ɛ2C_ɛ1 = 128°; FeN_ɛ2C_δ2= 127°; FeN_ɛ2N_δ1 = 163°; FeN_ɛ2C_γ= 162°; Methionine residue: Fe-S_δ = 2.29 Å; S_δ-C_γ = 1.81 Å; S_δ-C_ɛ = 1.82 Å; C_γ-C_β = 1.5 Å; FeS_δC_ɛ,γ = 109°; S_δC_γC_β = 110°.

FIGURE 2

Edge and enlarged preedge (inset) spectra for ferrocytochrome c in solution with glycerol.

FIGURE 3

Experimental k³-weighted XAFS signals for all measured samples (continuous lines). From the top to the bottom: protein in solution without (sol. 1) and with glycerol (sol. 2), protein embedded in the polyvinyl alcohol film (PVA), in the moderately dried trehalose matrix (trehal. 1), and in the extensively dried trehalose matrix (trehal. 2). The dashed lines represent calculated best fitting XAFS signals, corresponding to the parameters reported in Table 2. See text for details.

FIGURE 4

Simulations based on the reference structural model (see Fig. 1). The values of the DW factors have been fixed as in models 1 (dashed line) and 2 (continuous line) (see text for details). Together with the total XAFS signal (top), we show the main partial contributions.

FIGURE 5

Graphic representation of the porphyrin pyrrolic group fourth shell elongation in the extra dry trehalose matrix. To obtain a simple picture we maintained the planarity of the structure, keeping collinear the Fe and C_3,4 atoms in the elongation. With these assumptions the C_2,5-C_3,4 and the C₃-C₄ distances changed by 13% and 10%, respectively.