The fe2+ site of photosynthetic reaction centers probed by multiple scattering x-ray absorption fine structure spectroscopy: improving structure resolution in dry matrices

Abstract

We report on the x-ray absorption fine structure of the Fe(2+) site in photosynthetic reaction centers from Rhodobacter sphaeroides. Crystallographic studies show that Fe(2+) is ligated with four N(epsilon) atoms from four histidine (His) residues and two O(epsilon) atoms from a Glu residue. By considering multiple scattering contributions to the x-ray absorption fine structure function, we improved the structural resolution of the site: His residues were split into two groups, characterized by different Fe-N(epsilon) distances, and two distinct Fe-O(epsilon) bond lengths resolved. The effect of the environment was studied by embedding the reaction centers into a polyvinyl alcohol film and into a dehydrated trehalose matrix. Incorporation into trehalose caused elongation in one of the two Fe-N(epsilon) distances, and in one Fe-O(epsilon) bond length, compared with the polyvinyl alcohol film. The asymmetry detected in the cluster of His residues and its response to incorporation into trehalose are ascribed to the hydrogen bonds between two His residues and the quinone acceptors. The structural distortions observed in the trehalose matrix indicate a strong interaction between the reaction-centers surface and the water-trehalose matrix, which propagates deeply into the interior of the protein. The absence of matrix effects on the Debye-Waller factors is brought back to the static heterogeneity and rigidity of the ligand cluster.

FIGURE 1

Reference structural model of the Fe²⁺ ligand cluster. (Inset) The bending of the Glu residue, i.e., a rigid rotation by an angle Δα around an axis through O_ɛ1. The angle α was set to 94°, and the Fe²⁺-O_ɛ1 distance was set to 2.12 Å; the O_ɛ2 atom of Glu is thus placed at 2.34 Å from Fe²⁺. The two His residues in plane with the Glu residue are placed symmetrically, so that the angle between the two Fe²⁺-N_ɛ2 bonds of the two His, and the angles between each Fe²⁺-N_ɛ2 bond and the direction Fe²⁺-C_δ of the Glu, are equal (120°). The target Fe²⁺-N_ɛ2 distance for the His residues was 2.16 Å (see text for details). The bond lengths of the His imidazole group are given in ref. 45.

FIGURE 2

Experimental k³-weighted XAFS functions measured in RCs embedded into the PVA film (dots), best fit to the 2 + 2 His model (solid bold line), and the corresponding dominating contributions coming from single and multiple scattering. The values of best-fitting parameters are given in Table 1.

FIGURE 3

Experimental k³-weighted XAFS signal (dots), best fit to the 2 + 2 His model (solid bold line), and main contributions to the fit for RCs embedded into the dry trehalose matrix. The values of best-fitting parameters are given in Table 1.

FIGURE 4

Relationship between the Fe-O₁ and Fe-O₂ distances in bidentate coordination with Asp and Glu residues. Data shown as open circles were extracted from the MESPEUS database, currently under development at Edinburgh University (47), selecting XRD protein structures at resolutions higher than 1.5 Å. Solid symbols correspond to distances determined in the PVA (circles) and in the trehalose matrix (squares) in this study (see Table 1 and text for details). The labels O₁ and O₂ can be permuted, so that each carboxylate group is shown twice in the plot, which is symmetrical about the diagonal line, d(Fe-O₁) = d(Fe-O₂).