Cytochrome b6f
Created by Jordan Kramer
Cytochrome b6f (1VF5) from Mastigocladus laminosus is an integral membrane protein that mediates electron transfer between the photosystem II and photosystem I reaction centers by oxidizing lipophilic plastoquinol and reducing plastocyanin. The coupling of the oxidation-reduction and protonation-deprotonation is central to the mechanism of proton translocation in the complex (2). Cytochrome b6f generates a transmembrane electrochemical proton gradient for adenosine triphosphate synthesis in the thylakoid membrane of chloroplasts in plants, cyanobacteria, and green algae. The molecular weight of cytochrome b6f is 214,805.70 Da and its isoelectric point (pI) is 6.41.
The secondary structure of the cytochrome b6f complex is 35% alpha-helices, 34 beta-strands, and 70 segments showing bends. Cytochrome b6f exists in the chloroplast as a dimer with two identical proteins. Each monomer consists of four large subunits, including cytochrome f, cytochrome b6, the Rieske iron-sulfur protein (ISP), and subunit IV; as well as four small hydrophobic subunits, PetG, PetL, PetM, and PetN. The three-dimensional structures of the p-side, n-side, and transmembrane domains facilitate their roles in enzyme activity. The monomeric unit contains thirteen transmembrane helices: four in cytochrome b6 (helices A to D); three in subunit IV (helices E to G); and one each in cytochrome f, the ISP, and the four small hydrophobic subunits (1). The high frequency of alpha-helices in the transmembrane domain anchors the protein between the photosystem II and photosystem I reaction centers, enabling the coupling of sequential electron transfers. The two monomers form a protein-free central cavity on each side of the thylakoid membrane for the p-side and n-side domains. Cavity walls are formed by helices C,D, and F of one monomer, helices A and E of the other monomer, and the ISP transmembrane helix of either of the two. The cavity floor is formed by the N-terminal 25 residues of cytochrome b6 and by lipid head groups that fill the cavity in situ. The roof is formed by cd1 and cd2 p-side peripheral helices connecting helices C and D of cytochrome b6 and the C terminus of the ISP transmembrane helix. A small portal in the wall of each cavity is formed by helices C, cd1, and F, and leads to the Qp and Qn pocket on each side of the membrane. The Qp pocket is bounded by the [2Fe-2S] cluster, heme bp, and the “ef loop” connecting helices E and F of subunit IV (1). The Qn pocket is bounded by heme bn and heme x. The tight openings of the p-side and n-side cavities allow enough space for plastoquinol and plastoquinone entry.
The dimer interface is enriched in aromatic residues Phe52, Phe56, and Phe189 in the A and D helices of cytochrome b6. Cys35 on the n side of the A helix makes a single covalent thioether bond with heme x, placing the prosthetic group in close proximity to the center of the complex. Phe40, on the n side of the E helix, is parallel to heme x and near (6 to 9 Å) plastoquinone in the cavity (1). Invariant Tyr33, Gly38, Phe203, and Ile206 of cytochrome b6 and Phe40 and Ile44 of subunit IV contact heme x (2). The heme-binding site of each monomer is lined with Val26, Pro27, Pro28, His29, Asn31, Arg207, and Gln209 of cytochrome b6.
Cytochrome b6f has two primary functions: the oxidation of plastoquinol to a semiquinone and the reduction of plastocyanin. The process carried out by cytochrome b6f is called the Q cycle because of the activity of quinone variants. The Qp active site is the location for plastoquinol binding, resulting in oxidation and electron transfer. The Rieske iron-sulfur protein oxidizes the plastoquinol to a semiquinone and two protons are released to the thylakoid lumen. The reduced iron-sulfur protein transfers an electron through cytochrome f to plastocyanin. Transfer of the second electron from plastoquinol across the complex through two b hemes, or as anionic plastosemiquinone, and the resulting proton uptake from the electronegative side generate a proton electrochemical gradient across the membrane (1). Reduction of a plastoquinone to a plastosemiquinone takes place at the Qn site of the n-side central cavity. The binding of a second plastoquinol causes one electron to reduce oxidized plastocyanin in the high-potential electron transport chain. In the low-potential electron transport chain, the electron from heme bn is transferred to the semiquinone and the completely reduced plastoquinone receives two protons to form the doubly reduced plastoquinol.
Seven prosthetic groups per monomer of cytochrome b6f have been identified. One 2Fe-2S cluster, four hemes (one c-, two b-, and one x-type), one chlorophyll a (Chl), and one β-carotene were described per monomer (4). A heme is a prosthetic group that contains an iron atom in the center of a heterocyclic organic ring. Hemes bp and bn, in the core of the complex that is structurally conserved between bc1 and b6f complexes, bridge the second and fourth transmembrane helices of the cytochrome b polypeptide (3). The transfer of one electron from plastoquinol to the [2Fe-2S] cluster can generate a semiquinone radical that reduces the heme bp and initiates electron transfer through a low potential chain. The electron transfer from heme bp to heme bn on the stroma side of the complex allows heme bn to reduce plastoquinone-9 to a semiquinone. Heme x is bound to Cys35 by a single covalent thioether bond at a position between heme bn and the central cavity. Heme x does not appear to be required for Q cycle function because the other elements of the Q cycle (hemes bp and bn) are identically oriented in the b6f and bc1 complexes, have identical interheme distances, and have similar hydrophobic environments between hemes (1). Lack of activity in the Q cycle by heme x indicates a potential function as ferrodoxin-plastoquinone reductase, a reagent necessary for cyclic electron transfer. The positive surface potential of cytochrome b6 on the stromal side would facilitate docking of anionic ferredoxin to the n side of the complex near heme x. Heme cn acts a redox cofactor on cytochrome f and provides a binding site for tridecyl-stigmatellin, a p-side quinone analog inhibitor. The function of the chlorophyll a is unknown, but it may fill structure gaps, similarly to bound lipids in membrane proteins. A molecule of β-carotene occurs near the center of the transmembrane region between the helices of PetL and PetM. β-carotene is too far away from chlorophyll a for quenching of the excited triplet state, the presumed function of bound β-carotene (1).
Cytochrome bc1 (1BE3) has an approximate 84% sequence similarity to cytochrome b6f and a similar folding pattern. The results of DALI (Z=19.5) and protein BLAST (E=3e-46) searches show that cytochrome bc1 has both primary and tertiary similarity to cytochrome b6f. The DALI search is used for tertiary similarity and protein BLAST is used for primary structure. The intercofactor distances and the organization of 8 of the 13 transmembrane helices (A to D in cytochrome b6, E and F in subunit IV, ISP, and cytochrome f) are similar in the b6f and bc1 complexes, but access to the Qp and Qn sites within the cavity are different. Constriction of the portal by the chlorophyll phytyl tail causes the Qp site of cytochrome b6f to be less accessible than the Qp site of the bc1 complex (1). The Qn site in cytochrome b6f is more accessible because it is not an enclosed pocket. The two complexes have different Qn sites due to the additional Thr188 between the two histidine ligands in the D helix of cytochrome b6f and the introduction of heme x where the ubiquinone and antimycin A binding site would be in cytochrome bc1. Three prosthetic groups in the b6f complex, the heme cn, one chlorophyll a and one β-carotene molecule per b6f monomer, are uniquely found in the b6f complex, but not in the bc1 complex (3). The two protein complexes differ in the number of lipid binding sites; at least eleven lipids are associated with the bc1 complex and the b6f cytochrome contains approximately seventeen. One of these sites in the b6f complex is occupied by a natural sulfo-lipid which interacts with n-side segments of the ISP and cytochrome f (3). Cytochrome b6f and cytochrome bc1 carry out similar electron transport functions despite being located in the chloroplast and mitochondria respectively.
Cytochrome b6f (1Q90) from Chlamydomonas reinhardtii has an approximate 99% sequence similarity to cytochrome b6f of Mastigocladus laminosus and a similar folding pattern. The results of DALI (Z=26.4) and protein BLAST (E=7e-135) searches show that the cyt b6f of C. reinhardtii has greater primary and tertiary similarities with the cyt b6f of M. laminosus than were found with cytochrome bc1. The cytochrome b, subunit IV, PetG, PetL, PetM, and PetN subunits are common to both b6f cytochromes, but the Rieske iron-sulfur protein and cytochrome f have been replaced in C. reinhardtii. Apocytochrome f is a mutant version of cytochrome f with a 6 His tag at the C-terminus. Two iron-sulfur subunits are used in place of the Rieske iron-sulfur protein. The two iron-sulfur subunits contain a total of 176 amino acids while the Rieske iron-sulfur protein is comprised of 179, indicating a 3 residue difference that does not alter function. Three prosthetic groups in the C. reinhardtii complex, eicosane, 1,2-distearoyl-monoglactosyl-diglyceride, and sulfoquinovosyldiacylglycerol, are not found in the M. laminosus complex. Eicosane is necessary to increase stability of the n-side domain at the location of the two iron-sulfur subunits. A newly discovered feature of b-heme orientation in b6f complexes is that heme bp in M. laminosus is rotated 180o about the normal to the membrane plane relative to the heme orientation in Nostoc and Chlamydomonas reinhardtii (2). The rotation of heme bp in M. laminosus prevents obstruction of electron transfer from the anionic semiquinone. The cytochrome b6f complexes from M. laminosus and C. reinhardtii perform the same function despite minor structural differences.
An obstacle in the crystal structure studies has been proteolysis and resulting monomerization of the complex, which degrades the complex from its active dimer form. Proteolysis of cytochrome b6f in Mastigocladus laminosus occurs slowly enough that crystals can be obtained when the crystallization process is accelerated through addition of lipid to the purified complex. Addition of DOPC or DOPG lipid to the complex immediately after the last step of the purification process at a stoichiometry of 10:1, lipid:cytochrome f, resulted in formation of hexagonal crystals (3). Three lipid molecules, a sulfo-lipid and two DOPC molecules, can be resolved in the intermonomer quinone exchange cavity. Two additional natural lipids, MGDG, for a total of three lipids per monomer are required for crystallization of the C. reinhardtii complex (6). Presence of the lipids in the structure is not the only factor required to ensure dimer stability, since the dimeric b6f complex is retained prior to lipid augmentation. Trace amounts of protease could be destructive to the integrity of the dimer complex, necessitating a mixture of protease inhibitors in all solutions used for purification. Examination by SDS-PAGE of the M. laminosus b6f complex, which was kept in different detergents at room temperature for 7-14 days, showed proteolysis (3). The Rieske iron-sulfur protein suffered cleavage of a 48-amino acid C terminal fragment. The cyt b6f and subunit IV polypeptides were also clipped at their exposed termini. Proteolysis inhibitors at moderate concentration could retard but not inhibit proteolysis over a period of 1 week. The structure was solved by isomorphous replacement using Pb and Pt derivatives and multiwavelength anomalous diffraction from native iron atoms (1). Refinement of the model was carried out with a 3.0 Å data set from a second crystal with the quinone-analog inhibitor tridecyl-stigmatellin.
The genetic manipulation of cytochrome b6f composition is complicated by six of the eight subunits of the complex being plastid genome-encoded. The only subunits encoded in the nucleus are the Rieske iron-sulfur protein (encoded by the petC gene), the small peripheral PetM subunit, and the ferredoxin-NADP+ oxidoreductase encoded by the petH gene (5). The roles of the PetG, PetL, PetM, and PetN subunits are not known, but stabilizing activity is a possibility. The PetL subunit of cytochrome b6f is not essential for biogenesis and function, but removal in plant cells affected the mature leaves of tobacco plants. While young mutant leaves accumulate comparable amounts of cytochrome b6f complex and have an identical assimilation capacity as wild type leaves, both cytochrome b6f complex contents and assimilation capacities of mature and old leaves are strongly reduced in the mutant, indicating that the cytochrome b6f complex is less stable than in the wild type (5). Reduced complex stability was also confirmed by in vitro treatments of isolated thylakoids with chaotropic reagents.
Since light is not required for catalytic turnover of the cytochrome b6f complex, the role of the single chlorophyll a in the structure and function of the complex is unknown. The purified b6f complex from Tyr112Phe or Phe133Leu mutants was characterized by a loss of bound Chl and heme b, a shift in the absorbance peak and increase in bandwidth, and relatively small time-resolved absorbance anisotropy values of the Chl Qy band (7). The study was unable to determine the role of chlorophyll a, but it was concluded that the aromatic residues of subunit IV are important in maintaining the short lifetime of the singlet excited state which would normally undergo quenching from β-carotene. The probability of singlet oxygen formation decreases upon interaction with the cytochrome b6f complex.