Figure 1.
(Top) The heme (red) fits the FA1 site in subdomain IB. Sudlow's site I (in subdomain IIA, corresponding to FA7) is occupied by warfarin (magenta). Sudlow's site II (in subdomain IIIA, corresponding to FA3–FA4) and FA6 (in subdomain IIB) are occupied by ibuprofen (blue). Atomic coordinates were taken from PDB entries 1O9X [22], 2BXD [10], and 2BXG [10], picture drawn using the UCSF Chimera package [67]. (Bottom) The local heme-Fe(III) structure is highlighted, the atoms belonging to the heme-Fe(III) coordination shells are labeled.
Figure 2.
XANES data: comparison of Fe-K edge XANES measured on HSA-heme-Fe(III), ibuprofen-HSA-heme-Fe(III), and warfarin-HSA-heme-Fe(III).
The effect of drugs is mainly evident at the main XANES peak (arrow) while the edge position is largely unchanged, signaling the same Fe electronic state in HSA and drug added samples. All XAS data were collected at low temperature (around 20 K) and pH 7.0 (1.0×10−1 M phosphate buffer).
Figure 3.
Moduli of the Fourier transform of experimental (dots) and best fit (full line) k-weighted Fe-K edge EXAFS spectra of all the investigated samples (vertically shifted for sake of clarity).
The FT are prone to simple qualitative interpretation of the local structure around the absorber as the peaks denote a coordination shell. However, the phase shift function (Eq. 1) reduces the peak position by roughly 0.5 Å with respect to the real value.
Figure 4.
Experimental data (points) and best fit (full lines) for all the analyzed samples are shown. The partial contributions (shells) used for the refinement of each spectrum are shown (vertically shifted for clarity); the structural parameters are reported in Table 1. The residuals (experimental data minus best fit) are shown, at the bottom for each sample.
Table 1.
Local atomic structure of HSA-heme-Fe(III), ibuprofen-HSA-heme-Fe(III), and warfarin-HSA-heme-Fe(III) around the heme-Fe(III) atom as obtained by EXAFS data analysis, compared with the average coordination distances of models (PDB and SMD).
Figure 5.
Conformational transition of HSA-heme-Fe(III) upon ligand binding to the FA2 site.
Panel A. Three-dimensional representation of the starting crystal structure (cyan, PDB entry 1O9X [22]) of HSA-heme-Fe(III). Heme-Fe(III) and the His146 and Tyr161 residues are highlighted in blue. The Glu131-Arg145 α-helix is represented in magenta. Panel B. Three-dimensional representation of the final model (orange) of HSA-heme-Fe(III) obtained via SMDS. Heme-Fe(III) and the His146 and Tyr161 residues are highlighted in red. The Glu131-Arg145 α-helix is represented in green. Panel C. Superposition of the starting crystal structure and of the final model of HSA-heme-Fe(III). The picture has been drawn using the UCSF Chimera package [67], [68].
Figure 6.
Top curves represent the experimental XANES spectra of HSA-heme-Fe(III) (blue) and warfarin-HSA-heme-Fe(III) (red).
Middle curves are the XANES spectra calculated ab initio using the FEFF code [32] for atomic cluster made combining SMDS and EXAFS results. The bottom curves represent the density of d states (d-DoS). The calculated XANES spectra reproduce the main experimental features, in particular the inversion of B and B′ peaks upon drug binding is reproduced. The pre-edge peak A corresponds to the d-DoS.
Figure 7.
Semi-quantitative analysis of Fe(III) pre-edge peaks and comparison with reference compounds.
In the inset, the experimental Fe(III) pre-edge peak (red symbols) of HSA-heme-Fe(III) and warfarin-HSA-heme-Fe(III) are modelled combining an arctangent function (gray line), simulating the onset of the continuous electron states, and Gaussian peaks (blue lines), representing the transitions to localized electronic states. The grey ellipses enclose the values of Fe(III) pre-edge peak integrated areas versus their centroid positions, measured on tetra-, penta-, and hexa- coordinated Fe(III) reference compounds (labelled [4], [5], and [6], respectively; [56]). The pre-edge peak parameters of HSA-heme-Fe(III) (red circle) fall in the region of penta-coordinated Fe(III), while ibuprofen-HSA-heme-Fe(III) and warfarin-HSA-heme-Fe(III) pre-edge peaks parameters (blue circles) are in the region corresponding to hexa-coordinated Fe(III) compounds. The pre-edge fitting of drug-bound HSA-heme-Fe(III) samples requires an additional peak (labelled B) in the region about 2 eV above the main one (labelled A). Analogous components have been reported in ref. [54]. The size of red and blue symbols takes into account for best fit incertitude.