Atomic resolution structures of rieske iron-sulfur protein: role of hydrogen bonds in tuning the redox potential of iron-sulfur clusters

Abstract

The Rieske [2Fe-2S] iron-sulfur protein of cytochrome bc(1) functions as the initial electron acceptor in the rate-limiting step of the catalytic reaction. Prior studies have established roles for a number of conserved residues that hydrogen bond to ligands of the [2Fe-2S] cluster. We have constructed site-specific variants at two of these residues, measured their thermodynamic and functional properties, and determined atomic resolution X-ray crystal structures for the native protein at 1.2 A resolution and for five variants (Ser-154-->Ala, Ser-154-->Thr, Ser-154-->Cys, Tyr-156-->Phe, and Tyr-156-->Trp) to resolutions between 1.5 A and 1.1 A. These structures and complementary biophysical data provide a molecular framework for understanding the role hydrogen bonds to the cluster play in tuning thermodynamic properties, and hence the rate of this bioenergetic reaction. These studies provide a detailed structure-function dissection of the role of hydrogen bonds in tuning the redox potentials of [2Fe-2S] clusters.

Figure 1

Orthogonal views of the overall structure of the Rieske iron-sulfur protein from R. sphaeroides. (A) A ribbon representation of the polypeptide is shown labeled with the corresponding secondary structural elements as defined in the text. Atoms within the [2Fe-2S] cluster are shown as white and yellow spheres, respectively. (B) The structure is shown, rotated 90° about the vertical axis, viewed into the location of the [2Fe-2S] cluster.

Figure 2

(A–C) Difference Fourier maps shown around residue 156 of wild-type and variant Rieske ISP, calculated with coefficients F_o–F_c using experimental amplitudes and phases calculated from the final refined structures, minus the coordinates of residue 156. The contour levels of the maps are 2.5 σ (blue). Coordinates from the final refined structures are superimposed upon the respective maps, and atoms of the [2Fe-2S] cluster is shown as stick figures in magenta and orange. The structures shown are of (A) wild-type, (B) Tyr-156→Phe and (C) Tyr-156→Trp Rieske ISP. (D) Superposition of the structures of wild-type (cyan), and Tyr-156→Trp (yellow) ISP showing the gross structural movements that accompany the introduction of the Trp-156 side chain.

Figure 3

(A–D) Difference Fourier maps shown around residue 154 of wild-type and variant Rieske ISP, calculated with coefficients F_o–F_c using experimental amplitudes and phases calculated from the final refined structures, minus the coordinates of residue 154. The contour levels of the maps are 2.5 σ (blue), and 8 σ (red). Coordinates from the final refined structures are superimposed upon the respective maps and atoms of the [2Fe-2S] cluster is shown as stick figures in magenta and orange. The structures shown are of (A) wild-type, (B) Ser-154→Ala and (C) Ser-154→Cys, and (D) Ser-154→Thr Rieske ISP.