[Structure and function of the cytochrome b6f complex of oxygenic photosynthesis].
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Crystallography, X-Ray↗
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Ultrafast carotenoid-to-chlorophyll singlet energy transfer in the cytochrome b6f complex from Bryopsis corticulans.
Ultrafast carotenoid-to-chlorophyll (Car-to-Chl) singlet excitation energy transfer in the cytochrome b(6)f (Cyt b(6)f) complex from Bryopsis corticulans is investigated by the use of femtosecond time-resolved absorption spectroscopy. For all-trans-alpha-carotene free in n-hexane, the lifetimes of the two low-lying singlet excited states, S(1)(2A(g)(-)) and S(2)(1B(u)(+)), are determined to be 14.3 +/- 0.4 ps and 230 +/- 10 fs, respectively. For the Cyt b(6)f complex, to which 9-cis-alpha-carotene is bound, the lifetime of the S(1)(2A(g)(-)) state remains unchanged, whereas that of the S(2)(1B(u)(+)) state is significantly reduced. In addition, a decay-to-rise correlation between the excited-state dynamics of alpha-carotene and Chl a is clearly observed. This spectroscopic evidence proves that the S(2)(1B(u)(+)) state is able to transfer electronic excitations to the Q(x) state of Chl a, whereas the S(1)(2A(g)(-)) state remains inactive. The time constant and the partial efficiency of the energy transfer are determined to be 240 +/- 40 fs and (49 +/- 4)%, respectively, which supports the overall efficiency of 24% determined with steady-state fluorescence spectroscopy. A scheme of the alpha-carotene-to-Chl a singlet energy transfer is proposed based on the excited-state dynamics of the pigments.
Carotenoids↗
A new subunit of cytochrome b6f complex undergoes reversible phosphorylation upon state transition.
A 15.2-kDa polypeptide, encoded by the nuclear gene PETO, was identified as a novel cytochrome b(6)f subunit in Chlamydomonas reinhardtii. The PETO gene product is a bona fide subunit, subunit V, of the cytochrome b(6)f complex, because (i) it copurifies with the other cytochrome b(6)f subunits in the early stages of the purification procedure, (ii) it is deficient in cytochrome b(6)f mutants accumulating little of the complex, and (iii) it colocalizes with cytochrome f, which migrates between stacked and unstacked membrane regions upon state transition. Sequence analysis and biochemical characterization of subunit V shows that it has a one transmembrane alpha-helix topology with two large hydrophilic domains extending on the stromal and lumenal side of the thylakoid membranes, with a lumenal location of the N terminus. Subunit V is reversibly phosphorylated upon state transition, a unique feature that, together with its topological organization, points to the possible role of subunit V in signal transduction during redox-controlled short term and long term adaptation of the photosynthetic apparatus in eukaryotes.
Amino Acid Sequence↗
The inhibitor DBMIB provides insight into the functional architecture of the Qo site in the cytochrome b6f complex.
Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.
Binding Sites↗
Contrasted effects of inhibitors of cytochrome b6f complex on state transitions in Chlamydomonas reinhardtii: the role of Qo site occupancy in LHCII kinase activation.
We have investigated the relationship between the occupancy of the Q(o) site in the cytochrome b(6)f complex and the activation of the LHCII protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII proteins, while subunit V of the cytochrome b(6)f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b(6)f that relates the activation of the LHCII kinase to the occupancy of the Q(o) site and the movement of the Rieske protein.
Animals↗
PetC1 is the major Rieske iron-sulfur protein in the cytochrome b6f complex of Synechocystis sp. PCC 6803.
Many of the completely sequenced cyanobacterial genomes contain a gene family that encodes for putative Rieske iron-sulfur proteins. The Rieske protein is one of the large subunits of the cytochrome bc-type complexes involved in respiratory and photosynthetic electron transfer. In contrast to all other subunits of this complex that are encoded by single genes, the genome of the cyanobacterium Synechocystis PCC 6803 contains three petC genes, all encoding potential Rieske subunits. Most interestingly, any of the petC genes can be deleted individually without altering the Synechocystis phenotype dramatically. In contrast, double deletion experiments revealed that petC1 and petC2 cannot be deleted in combination, whereas petC3 can be deleted together with any of the other two petC genes. Further results suggest a different physiological function for each of the Rieske proteins. Whereas PetC2 can partly replace the dominating Rieske isoform PetC1, PetC3 is unable to functionally replace either PetC1 or PetC2 and may have a special function involving a special donor with a lower redox potential than plastoquinone. A predominant role of PetC1, which is (partly) different from PetC2, is suggested by the mutational analysis and a detailed characterization of the electron transfer reactions in the mutant strains.
Chlorophyll↗
Identification of two 4-kDa miniproteins in the cytochrome b6f complex from Chlamydomonas reinhardtii.
Two low molecular weight subunits (ca. 4 kDa) have been identified in highly purified preparations of cytochrome b6f from the green unicellular alga Chlamydomonas reinhardtii. The N-terminus of the first one is blocked. It is synthesized in the chloroplast. It cross reacts with an antiserum raised against a synthetic peptide with the C-terminal sequence of the predicted product of the chloroplast gene petG. The homologue of this protein had been previously identified by other authors in the b6f complex from maize. The N-terminal sequence of the second subunit does not correspond to any known protein. This polypeptide, provisionally named petX, is synthesized in the cytosol. An antiserum has been raised against the corresponding synthetic peptide. Immunoblotting experiments show that neither petG nor petX are present in thylakoid membranes from a b6f-less strain of C. reinhardtii.
Animals↗
Ssr2998 of Synechocystis sp. PCC 6803 is involved in regulation of cyanobacterial electron transport and associated with the cytochrome b6f complex.
To analyze the function of a protein encoded by the open reading frame ssr2998 in Synechocystis sp. PCC 6803, the corresponding gene was disrupted, and the generated mutant strain was analyzed. Loss of the 7.2-kDa protein severely reduced the growth of Synechocystis, especially under high light conditions, and appeared to impair the function of the cytochrome b6 f complex. This resulted in slower electron donation to cytochrome f and photosystem 1 and, concomitantly, over-reduction of the plastoquinone pool, which in turn had an impact on the photosystem 1 to photosystem 2 stoichiometry and state transition. Furthermore, a 7.2-kDa protein, encoded by the open reading frame ssr2998, was co-isolated with the cytochrome b6 f complex from the cyanobacterium Synechocystis sp. PCC 6803. ssr2998 seems to be structurally and functionally associated with the cytochrome b6 f complex from Synechocystis, and the protein could be involved in regulation of electron transfer processes in Synechocystis sp. PCC 6803.
Amino Acid Sequence↗
Conformational changes in the cytochrome b6f complex induced by inhibitor binding.
Binding of stigmatellin, an inhibitor of the Q(o) site of the bc-type complexes, has been shown to induce large conformational changes of the Rieske protein in the respiratory bc(1) complex (Kim, H., Xia, D., Yu, C. A., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8026-8033; Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., Link, T. A., Ramaswamy, S., and Jap, B. K. (1998) Science 281, 64-71; Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y. I., Kim, K. K., Hung, L. W., Crofts, A. R., Berry, E. A., and Kim, S. H. (1998) Nature 392, 677-684). Such a movement seems necessary to shuttle electrons from the membrane-soluble quinol to the extramembrane heme of cytochrome c(1). To see whether similar changes occur in the related photosynthetic b(6)f complex, we have studied the effect of the binding of stigmatellin to the eukaryotic b(6)f complex by electron crystallography. Comparison of projection maps of thin three-dimensional crystals prepared with or without stigmatellin, and either negatively stained or embedded in glucose, reveals a similar type of movement to that observed in the bc(1) complex and suggests also the occurrence of conformational changes in the transmembrane region.
Animals↗
The pgr1 mutation in the Rieske subunit of the cytochrome b6f complex does not affect PGR5-dependent cyclic electron transport around photosystem I.
Although photosystem I (PSI) cyclic electron transport is essential for plants, our knowledge of the route taken by electrons is very limited. To assess whether ferredoxin (Fd) donates electrons directly to plastoquinone (PQ) or via a Q-cycle in the cytochrome (cyt) b(6)f complex in PSI cyclic electron transport, we characterized the activity of PSI cyclic electron transport in an Arabidopsis mutant, pgr1 (proton gradient regulation). In pgr1, Q-cycle activity was hypersensitive to acidification of the thylakoid lumen because of an amino acid alteration in the Rieske subunit of the cyt b(6)f complex, resulting in a conditional defect in Q-cycle activity. In vitro assays using ruptured chloroplasts did not show any difference in the activity of PGR5-dependent PQ reduction by Fd, which functions in PSI cyclic electron transport in vivo. In contrast to the pgr5 defect, the pgr1 defect did not show any synergistic effect on the quantum yield of photosystem II in crr2-2, a mutant in which NDH (NAD(P)H dehydrogenase) activity was impaired. Furthermore, the simultaneous determination of the quantum yields of both photosystems indicated that the ratio of linear and PSI cyclic electron transport was not significantly affected in pgr1. All the results indicated that the pgr1 mutation did not affect PGR5-dependent PQ reduction by Fd. The phenotypic differences between pgr1 and pgr5 indicate that maintenance of the proper balance of linear and PSI cyclic electron transport is essential for preventing over-reduction of the stroma.
Arabidopsis↗
Heterogeneous Rieske proteins in the cytochrome b6f complex of Synechocystis PCC6803?
The completely sequenced genome of the cyanobacterium Synechocystis PCC6803 contains three open reading frames, petC1, petC2, and petC3, encoding putative Rieske iron-sulfur proteins. After heterologous overexpression, all three gene products have been characterized and shown to be Rieske proteins as typified by sequence analysis and EPR spectroscopy. Two of the overproduced proteins contained already incorporated iron-sulfur clusters, whereas the third one formed unstable aggregates, in which the FeS cluster had to be reconstituted after refolding of the denatured protein. Although EPR spectroscopy showed typical FeS signals for all Rieske proteins, an unusual low midpoint potential was revealed for PetC3 by EPR redox titration. Detailed characterization of Synechocystis membranes indicated that all three Rieske proteins are expressed under physiological conditions. Both for PetC1 and PetC3 the association with the thylakoid membrane was shown, and both could be identified, although in different amounts, in the isolated cytochrome b(6)f complex. The considerably lower redox potential determined for PetC3 indicates heterogeneous cytochrome b(6)f complexes in Synechocystis and suggests still to be established alternative electron transport routes.
Amino Acid Sequence↗
Kinetic effects of the electrochemical proton gradient on plastoquinone reduction at the Qi site of the cytochrome b6f complex.
We have investigated the effects of the light-induced thylakoid transmembrane potential on the turnover of the b(6)f complex in cells of the unicellular green alga Chlamydomonas reinhardtii. The reduction of the potential by either decreasing the light intensity or by adding increasing concentrations of the ionophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) revealed a marked inhibition of the cytochrome b(6) oxidation rate (10-fold) without substantial modifications of cytochrome f oxidation kinetics. Partial recovery of this inhibition could be obtained in the presence of ionophores provided that the membrane potential was re-established by illumination with a train of actinic flashes fired at a frequency higher than its decay. Measurements of isotopic effects on the kinetics of cytochrome b(6) oxidation revealed a synergy between the effects of ionophores and the H(2)O-D(2)O exchange. We propose therefore, that protonation events influence the kinetics of cytochrome b(6) oxidation at the Qi site and that these reactions are strongly influenced by the light-dependent generation of a transmembrane potential.
Animals↗
Solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus.
Cytochrome c6 is a small, soluble electron carrier between the two membrane-bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis. We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints. The overall fold exhibits four alpha-helices and a small antiparallel beta-sheet in the vicinity of Met58, one of the axial heme ligands. The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants. This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex. The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI. This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S. elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f. As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.
Amino Acid Sequence↗
An engineered cytochrome b6c1 complex with a split cytochrome b is able to support photosynthetic growth of Rhodobacter capsulatus.
The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.
Cytochrome b Group↗
Characterization and crystallization of the lumen side domain of the chloroplast Rieske iron-sulfur protein.
A soluble, 139-residue COOH-terminal polypeptide fragment of the Rieske iron-sulfur protein of the cytochrome b6f complex from spinach chloroplasts was obtained by limited proteolysis of the complex and a two-step chromatography purification protocol. The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 +/- 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized optical difference spectrum characterized by DeltaepsilonmM = 3.8 mM-1 cm-1 for DeltaA at 394 versus 409 nm, which was used to determine the midpoint oxidation-reduction potential, which is +359 +/- 7 mV at 25 degrees C from pH 5.5-6.5, and +319 +/- 2 mV at pH 7, with an apparent pKox = 6.5 +/- 0.2 for the oxidized protein. The EPR spectrum measured at 17 K was characterized by the g values, gz = 2.03 and gy = 1.90, and a broad band centered at gx approximately 1.74, very similar or identical to those of the Rieske cluster in the b6f complex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex. Midpoint potential determination by low temperature EPR yielded a redox midpoint potential (Em) of +365-375 mV of the soluble Rieske fragment at pH 6 and 7 and an Em of +295-300 mV of the Rieske cluster in the cytochrome b6f complex at pH 6 and 7. The Em difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluster in the intact complex. Single crystals of the Rieske polypeptide were obtained that are capable of x-ray diffraction to atomic resolution (<2.5 A), contain one molecule per asymmetric unit, a solvent content of approximately 30%, and belong to the triclinic space group P1 with cell dimensions, a = 29.1 A, b = 31.9 A, c = 35.8 A, alpha = 95.6 degrees, beta = 107.1 degrees, gamma = 117.3 degrees.
Amino Acid Sequence↗
Quinol-cytochrome c oxidoreductase from the thermophilic bacterium PS3. Purification and properties of a cytochrome bc1(b6f) complex.
A quinol-cytochrome c oxidoreductase (cytochrome bc1 complex) has been purified from plasma membranes of a thermophilic Bacillus, PS3, by ion-exchange chromatography in the presence of Triton X-100. The purified enzyme shows absorption bands at 561-562 nm and 553 nm at room temperature, and 560, 551, and 547 nm at 80 K upon reduction, and gives an ESR signal similar to that of a Rieske-type iron sulfur center. Its contents of protohemes, heme c, and non-heme iron are about 23, 10, and 21 nmol/mg of protein, respectively. The enzyme consists of four polypeptides with molecular masses of 29, 23, 21, and 14 kDa judging from their electrophoretic mobilities in the presence of sodium lauryl sulfate. Since the staining intensities of the respective bands are almost proportional to their molecular masses, the monomer complex (87 kDa) of the subunits probably consists of a cytochrome b having two protohemes, a cytochrome c1 and an Fe2-S2-type iron sulfur center. The 29 and 21 kDa subunits were identified as cytochromes c1 and b, respectively, and the 23-kDa subunit is probably an iron-sulfur protein, since the 14-kDa polypeptide can be removed with 3 M urea without reducing the content of non-heme iron. Several characteristics of the subunits and chromophores indicate that the PS3 enzyme is rather similar to cytochrome b6f (a bc1 complex equivalent) of chloroplasts and Cyanobacteria. The PS3 complex catalyzes reduction of cytochrome c with various quinol compounds in the presence of P-lipids and menaquinone. The turnover number at pH 6.8 was about 5 s-1 at 40 degrees C and 50 s-1 at 60 degrees C. The enzyme is heat-stable up to 65 degrees C.
Bacillus↗
Evidence for a cytochrome f-Rieske protein subcomplex in the cytochrome b6f system from spinach chloroplasts.
The cytochrome b6f complex of spinach chloroplasts was prepared with minor modification according to the method of E. Hurt and G. Hauska (1981) Eur. J. Biochem. 117, 591-599) replacing, however, the final ultracentrifugation step by hydroxyapatite chromatography as suggested by M. F. Doyle and C.-A Yu (1985) Biochem. Biophys. Res. Commun. 131, 700-706). The purified complex was partially dissociated by treatment with 4 M urea or 0.1% sodium dodecyl sulfate (SDS) in the absence of reducing agents. A binary subcomplex consisting of cytochrome f and the Rieske iron-sulfur protein was observed under these conditions by three different methods: (a) hydroxyapatite chromatography; (b) extraction with an isopropanol/water/trifluoroacetic acid mixture; and (c) gel filtration in the presence of low SDS concentrations. The subcomplex dissociated into its components by treatment with mercaptoethanol. These results suggest a close interaction of the cytochrome f with the Rieske protein involving SH groups which under reducing conditions leads to complete dissociation of the subcomplex.
Chlorophyll↗
The chloroplast Rieske iron-sulfur protein. At the crossroad of electron transport and signal transduction.
We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur protein; that is, the flexible hinge that connects the transmembrane helix to the soluble cluster-bearing domain, the N-terminal stromal protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b6f complex, perform plastoquinol oxidation, and signal redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo. Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency. Deletion of three residues (Delta3G) dramatically decreased reaction rates by a factor of approximately 10. These data indicate that the chloroplast iron-sulfur protein-linking domain is much more flexible than that of its counterpart in mitochondria. Despite greatly slowed catalysis in the Delta3G mutant, there was no apparent delay in light-harvesting complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske protein did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske protein transmembrane helix resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske protein transmembrane helix plays an active role in assembly-mediated control of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b6f structure.
Amino Acid Sequence↗