Lipid-induced conformational changes within the cytochrome b6f complex of oxygenic photosynthesis

Abstract

Cytochrome b6f catalyzes quinone redox reactions within photosynthetic membranes to generate a transmembrane proton electrochemical gradient for ATP synthesis. A key step involves the transfer of an electron from the [2Fe-2S] cluster of the iron-sulfur protein (ISP) extrinsic domain to the cytochrome f heme across a distance of 26 Å, which is too large for competent electron transfer but could be bridged by translation-rotation of the ISP. Here we report the first crystallographic evidence of significant motion of the ISP extrinsic domain. It is inferred that extensive crystallographic disorder of the ISP extrinsic domain indicates conformational flexibility. The ISP disorder observed in this structure, in contrast to the largely ordered ISP structure observed in the b6f complex supplemented with neutral lipids, is attributed to electrostatic interactions arising from anionic lipids.

Fig 1

Cyt b₆f complex from the moderately thermophilic cyanobacterium Mastigocladus laminosus (PDB ID 2E74). Polypeptide subunits are shown as ribbons and prosthetic groups as sticks and spheres. Color scheme: Cytochrome b₆ (cyt b₆), pink; subunit IV (sub IV), cyan; cytochrome f (cyt f), wheat; Iron-Sulfur Protein (ISP), orange; Pet G, brown; Pet L, gray; Pet M, yellow; Pet N, green.

Fig 2

p-Side extrinsic domain architecture of the cyt b₆f (PDB ID 2E74) and bc₁ complex (PDB ID 3CX5). (A, B) The cyt f extrinsic domain (yellow) consists of an elongated β–sheet structure that extends over 75 Ål. (C, D) In contrast, the cyt c₁ extrinsic domain (yellow) is predominantly α–helical in structure and is more compact. The ISP polypeptide is shown in pale green, the heme of cyt f/cyt c₁ in green/red (sticks) and the ISP [2Fe-2S] cluster as brown/wheat spheres.

Fig 3

Docking interface for ISP extrinsic domain. The figure was generated by superposition of three crystal structures of the cyt b₆f complex- PDB IDs 2E74, 2E76 and 4I7Z (described in present study). Residues from cyt b₆ are identified as “A” and those of sub IV by “B”. The ISP extrinsic domain is shown as wheat colored ribbon and includes the [2Fe-2S] cluster. Residues are color coded according to their respective crystal structures- PDB ID 2E74 (yellow), PDB ID 2E76 (cyan) and PDB ID 4I7Z (green). No large conformational changes are observed in the ISP docking interface.

Fig 4

Anionic lipid binding sites in cyt b₆f. (A) Four anionic lipid sites in the b₆f complex. (B) PG1 bound between the F and G TMH of sub IV. (C) PG2 inserted between peripheral Pet subunits. (D) PG3 located between ISP and cyt f TMH. Mesh, 2Fo-Fc map, 1.2 σ. Color code same as in Fig. 1.

Fig 5

Lipid-dependence of stability of purified cyt b₆f complex in lipids. Purified cyt b₆f complex from spinach thylakoid membranes was reconstituted with lipids (DOPG or DOPC) at a molar ratio (protein:lipid) of 1:10. The molecular mass of the ISP subunit does not undergo a change in the presence of the anionic lipid DOPG thereby indicating that the polypeptide remains intact in the presence of the anionic lipid. The band for the enzyme FNR, which co-purifies with the spinach cyt b₆f complex, is also shown.