Quinone-dependent proton transfer pathways in the photosynthetic cytochrome b6f complex

Abstract

As much as two-thirds of the proton gradient used for transmembrane free energy storage in oxygenic photosynthesis is generated by the cytochrome b6f complex. The proton uptake pathway from the electrochemically negative (n) aqueous phase to the n-side quinone binding site of the complex, and a probable route for proton exit to the positive phase resulting from quinol oxidation, are defined in a 2.70-Å crystal structure and in structures with quinone analog inhibitors at 3.07 Å (tridecyl-stigmatellin) and 3.25-Å (2-nonyl-4-hydroxyquinoline N-oxide) resolution. The simplest n-side proton pathway extends from the aqueous phase via Asp20 and Arg207 (cytochrome b6 subunit) to quinone bound axially to heme c(n). On the positive side, the heme-proximal Glu78 (subunit IV), which accepts protons from plastosemiquinone, defines a route for H(+) transfer to the aqueous phase. These pathways provide a structure-based description of the quinone-mediated proton transfer responsible for generation of the transmembrane electrochemical potential gradient in oxygenic photosynthesis.

Fig. 1.

Dimeric cytochrome b₆f complex of oxygenic photosynthesis from the cyanobacterium Nostoc PCC 7120. (A) Eight transmembrane polypeptides form the cytochrome b₆f monomer. Cytochrome b₆ (cyt b₆, cyan) and subunit IV (SubIV, pink) with four and three transmembranes helices, respectively, are polytopic subunits. Cytochrome f (cyt f, yellow) and the Rieske ISP (orange) have one transmembrane helix and a large soluble domain. The Pet G (brown), L (wheat), M (green), and N (blue) are located on the periphery of the complex and consist of one transmembrane helix and no soluble domains. p- and n- side, electrochemically positive and negative side of the membrane. (B) Prosthetic groups in the b₆f complex that function in electron transfer. The transmembrane pathway of electron transfer consists of 2 bis-histidine ligated hemes (b_p and b_n) and one covalently linked heme c_n, all linked to the cytochrome b₆ subunit. The p-side soluble domain of the ISP subunit has an iron-sulfur cluster ([2Fe-2S]), and the cytochrome f soluble domain has a covalently attached heme (f). n- and p-side proton transfer pathways are highlighted to show their position in the membrane. The n-side pathway consists of Asp20 (cyan and red sticks) and Arg207 (cyan and blue sticks) of cytochrome b₆. The p-side water channel is shown in green extending from the Glu78 residue (pink and red sticks) of subunit IV. The lipid bilayer has been modeled in cartoon mode (orange).

Fig. 2.

N-side proton uptake pathways of the cytochrome b₆f complex. (A) The D/R proton uptake pathway in the 2.70-Å Nostoc PCC 7120 cytochrome b₆f structure. The Asp20→Arg207 route of proton transfer is demonstrated. The Arg207/heme c_n interaction is also shown. Orange mesh, 2Fo-Fc map, 1.3 σ. (B and C) Arg207 as a ligand to the quinone analog inhibitors TDS (B) and NQNO (C) bound at the Q_n-site. From the cocrystal structures of b₆f with quinone analog inhibitors, it is inferred that the Arg207 side chain interacts with the natural quinone bound at the Q_n-site. Polypeptide color code, same as in Fig. 1, Heme b_p, brown lines; heme c_n, blue lines; TDS and NQNO, green red and blue sticks; 2Fo-Fc map, 1.2 σ, orange mesh. The peripheral Pet G, L, M, and N subunits and the hydrocarbon tails of TDS and NQNO are not shown. (D) Potential proton uptake pathway mediated by water in the b₆f complex. Water416 (red sphere) is coordinated by the Arg207 backbone carbonyl oxygen (a = 2.8 Å) and side chain (b = 3.3 Å), along with the propionate-A (c = 2.7 Å) carboxylic acid group of heme c_n (blue) (Fig. S2 shows heme nomenclature). Wat416 is separated from the basic side chain of Lys24 (cytochrome b₆, cyan) by a distance of 4.6 Å. The ISP transmembrane helix is omitted. (E and F) The E/D pathway of proton entry on the n-side of the b₆f complex. (E) In the 2.70-Å structure, Glu29 (subunit IV) located on the n-side surface of cytochrome b₆f interacts with a water molecule (“Wat,” red sphere), which forms a hydrogen bond with the acidic carboxylate side chain of Asp35 (subunit IV). (F) The Asp35 residue interacts with the quinone analog TDS bound at the Q_n-site. 2Fo-Fc map, 1.2 σ, orange mesh. For simplicity, the TDS hydrocarbon tail is not shown. Color code, same as in Fig. 1. Transmembrane helices of cyt b₆ (a–d) and subunit IV (e–g) are labeled.

Fig. 3.

Proposed proton exit pathways on the p-side of the cyt b₆f complex. A hydrophilic portal (green surface) leads from Glu78 of subunit IV to the p-side surface, providing a route for diffusion of water. The portal is lined by Arg87 (cytochrome b₆), Ser91 (cytochrome b₆), Glu3 (PetG), and Asp58 (subunit IV). Glu78 is linked to water 316 (wat316 in the figure) through a hydrogen bond that lies at the end of this portal. The proton exit pathway is highlighted through the hydrophilic portal (green) by a broken arrow (black). The TDS quinone analog inhibitor has been introduced from PDB ID 2E76 to mark the position of the Q_p-site. Orange mesh, 2Fo-Fc map, 1.3 σ. Transmembrane helices of cytochrome b₆ (a–d) and subunit IV (e–g) are labeled.