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Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex

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Quinone-Dependent Proton Transfer Pathways in the Photosynthetic Cytochrome B6f Complex Quinone-dependent proton transfer pathways in the photosynthetic cytochrome b6f complex S. Saif Hasana, Eiki Yamashitab, Danas Baniulisc, and William A. Cramera,1 aDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907; bInstitute for Protein Research, Osaka University, Osaka 565-0871, Japan; and cInstitute of Horticulture, Lithuanian Research Center for Agriculture and Forestry, Babtai LT-54333, Lithuania Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved February 1, 2013 (received for review December 19, 2012) As much as two-thirds of the proton gradient used for transmem- and the eukaryotic green alga Chlamydomonas reinhardtii (PDB brane free energy storage in oxygenic photosynthesis is generated ID 1Q90) (25). The substrate quinone undergoes reduction and by the cytochrome b6f complex. The proton uptake pathway from protonation at the n-side quinone (Qn) binding site of cyto- the electrochemically negative (n) aqueous phase to the n-side qui- chrome bc complexes. The unique heme cn at the n-side quinone none binding site of the complex, and a probable route for proton (Qn) binding site of the b6f complex (22, 26), located in close (4 Å) exit to the positive phase resulting from quinol oxidation, are de- proximity to heme bn, is a major structure difference between the fined in a 2.70-Å crystal structure and in structures with quinone bc1 and b6f complexes [n.b., heme cn is designated heme ci in analog inhibitors at 3.07 Å (tridecyl-stigmatellin) and 3.25-Å the C. reinhardtii structure (25)]. Heme cn has no axial ligand on (2-nonyl-4-hydroxyquinoline N-oxide) resolution. The simplest n-side the heme surface facing the intermonomer cavity. As shown by proton pathway extends from the aqueous phase via Asp20 and electron paramagnetic resonance (EPR) studies that document Arg207 (cytochrome b6 subunit) to quinone bound axially to heme its open axial ligand position (27), and cocrystallization of the b6f cn. On the positive side, the heme-proximal Glu78 (subunit IV), complex with quinone analog inhibitors (22), it has been inferred which accepts protons from plastosemiquinone, defines a route that heme cn serves as the n-side quinone binding site (Qn-site). + for H transfer to the aqueous phase. These pathways provide A consequence of this motif is that the Qn-site is located closer to – a structure-based description of the quinone-mediated proton the membrane water interface in the b6f complex than in the transfer responsible for generation of the transmembrane electro- respiratory bc1 complex, as noted in the original crystal structures chemical potential gradient in oxygenic photosynthesis. of the C. reinhardtii b6f complex (25). This surface-proximal lo- cation of the Qn-site implies that a short pathway may suffice for membrane protein | transmembrance electrochemical gradient | respiration proton conduction to the n-side bound quinone from the n-side aqueous phase. Before the present study, with a more limited resolution (3.0–3.8 Å) (21–25) of the cytochrome b f crystal he quantitative nature of the experimental tools used for 6 structures, it was not possible to identify the amino acids and analysis of electron and proton transfer function in energy- T water molecules that define the proton transfer pathways in transducing proteins allow precise analyses of function, which led the complex. The 2.70-Å crystal structure of the b f complex to these proteins being the subject of first crystal structures of 6 reported in the present study (Table 1), obtained from the fila- membrane proteins (1, 2). A detailed description of the structure mentous cyanobacterium Nostoc PCC 7120, revealed a unique of the proton-pumping pathways in these proteins followed the anhydrous Asp20→Arg207 (cytochrome b ) pathway for proton application of site-directed mutagenesis to problems of mem- 6 conduction from the n-side aqueous phase to the Q -site for brane protein structure-function, and of the improvement of n reduction and quinone protonation (Fig. 1B). Although the structure resolution that allowed detection of intraprotein water presence of water molecules in the n-side pathway was predicted molecules and water chains (3). The two mechanisms known for (19), the simpler Asp20→Arg207 pathway is devoid of water generation of the electrochemical proton gradient coupled to molecules. An alternate n-side Glu29→Asp35 (subunit IV) electron transport chains are (i) coupling of proton translocation BIOPHYSICS AND proton transfer pathway contains a water molecule. The exit to electron transfer, as in cytochrome oxidase (4–8), and (ii) pathway (Fig. 1B) for the second proton from the p-side quinol COMPUTATIONAL BIOLOGY oxidation-reduction of the lipophilic quinol proton carrier in binding (Q ) site is significantly hydrated. cytochrome bc complexes (9), which include the photosynthetic p b6f and the respiratory and bacterial cytochrome bc1 complexes. Results The pathway of proton transfer coupled to electron transport A highly conserved Asp20 residue (Fig. S1) is located on the ex- has been described most completely in crystal structures of the posed N terminus of the cytochrome b polypeptide (Figs. 1B and bacterial photosynthetic reaction center (3) and cytochrome 6 2A). In the 2.70-Å crystal structure of the b f complex described in oxidase (4, 5, 8), and less completely in the mitochondrial (10– 6 the present study, the acidic side chain of Asp20 interacts with the 13) and photosynthetic bacterial (14–17) bc complex. Water 1 basic guanidinium side chain of Arg207 (Fig. 2A) located on the molecules have been found to be ubiquitous in the solved proton n-side of the cytochrome b D-helix through a 3.4-Å hydrogen transfer networks of these energy-transducing proteins. Neither 6 bonding distance that is conducive to proton transfer between the the composition nor the structure of the proton transfer chains has been solved in the b6f complex. On the basis of structure analogy with the bc1 complex (18) and the effect of mutagenesis on redox properties, it has been inferred that on the negative (n)- Author contributions: W.A.C. designed research; S.S.H., E.Y., and D.B. performed research; W.A.C. contributed new reagents/analytic tools; S.S.H., E.Y., D.B., and W.A.C. analyzed side, Arg207 of cytochrome b6 (Arg214 in Synechococcous PCC data; and S.S.H. and W.A.C. wrote the paper. 7002) (19) and on the positive (p)-side, Glu78 (20) in the con- The authors declare no conflict of interest. served Pro-Glu-Trp-Tyr (PEWY) sequence of subunit IV, are This article is a PNAS Direct Submission. involved in pathways of proton transfer. Data deposition: The atomic coordinates and structure factors have been deposited in the Crystal structures of the cytochrome b6f complex (Fig. 1 A and Protein Data Bank, www.pdb.org [PDB ID codes 4H44 (native cyt b6f structure), 4H13 B) obtained in the native state, and in the presence of different (tridecyl-stigmatellin–containing structure), and 4H0L (2-nonyl-4-hydroxyquinoline quinone analog inhibitors, have been obtained from the pro- N-oxide–containing structure)]. karyotic filamentous cyanobacteria Mastigocladus laminosus 1To whom correspondence should be addressed. E-mail: [email protected]. [Protein Data Bank (PDB) IDs 1VF5, 2D2C, 2E74, 2E75, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2E76] (21–23) and Nostoc sp. PCC 7120 (PDB ID 2ZT9) (24), 1073/pnas.1222248110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1222248110 PNAS | March 12, 2013 | vol. 110 | no. 11 | 4297–4302 Downloaded by guest on September 27, 2021 b6f complex at both the Qp and Qn-quinone binding sites (22), whereas NQNO binds exclusively at the Qn-site of the b6f complex (22, 28). The Arg207 side chain guanidinium group (nitrogen atom labeled NH1) interacts with the TDS molecule (oxygen atom labeled OAC) through a 2.9-Å hydrogen bond (Fig. 2B).The in- teraction between Asp20 (OD2 atom of side chain carboxylate) and Arg207 (NH2 atom of the side chain guanidinium group), which forms the first half of the n-side proton conduction chain, occurs through a 3.0-Å bond. The role of Arg207 as a ligand to the quinone bound at the Qn-site is supported by a 2.6-Å H-bond between the NH1 atom of the Arg207 side chain guanidinium group and the O41 atom of the NQNO molecule bound at the Qn- site (Fig. 2C). The Asp20-Arg207 side chain interaction in the NQNO bound structure of the b6f complex also occurs through an H-bond of 2.9 Å. Therefore, this n-side proton conducting path- way consists of two steps: transfer from Asp20 to Arg207 and from Arg207 to quinone, both mediated by hydrogen bonds. Protons can be readily transferred across these distances without the in- troduction of any connecting water molecules. The Arg207 side chain interacts not only with quinone bound at the Qn-site but also with the heme cn propionate (Fig. 2A), as previously suggested (25). A 2.8-Å bond links the basic side chain of Arg207 and the O1D atom of the acidic carboxylate group of propionate-D (heme nomenclature in Fig. S2). Therefore, Arg207 has access to the Qn- site quinone as well as heme cn. The pH-dependence of the heme cn midpoint redox potential observed in the b6f complex from C. reinhardtii (26) could be attributed to its interaction with Arg207. More than one pathway of proton transfer may be functional on the n-side of the b6f complex (29). One possible additional pathway consists of a water molecule, wat416 (chain A), bound on the n-side of heme cn and the Arg207 side chain (Fig.
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