On rate limitations of electron transfer in the photosynthetic cytochrome b6f complex

Abstract

Considering information in the crystal structures of the cytochrome b(6)f complex relevant to the rate-limiting step in oxygenic photosynthesis, it is enigmatic that electron transport in the complex is not limited by the large distance, approximately 26 Å, between the iron-sulfur cluster (ISP) and its electron acceptor, cytochrome f. This enigma has been explained for the respiratory bc(1) complex by a crystal structure with a greatly shortened cluster-heme c(1) distance, leading to a concept of ISP dynamics in which the ISP soluble domain undergoes a translation-rotation conformation change and oscillates between positions relatively close to the cyt c(1) heme and a membrane-proximal position close to the ubiquinol electron-proton donor. Comparison of cytochrome b(6)f structures shows a variation in cytochrome f heme position that suggests the possibility of flexibility and motion of the extended cytochrome f structure that could entail a transient decrease in cluster-heme f distance. The dependence of cyt f turnover on lumen viscosity is consistent with a role of ISP - cyt f dynamics in determination of the rate-limiting step under conditions of low light intensity. Under conditions of low light intensity and proton electrochemical gradient present, for example, under a leaf canopy, it is proposed that a rate limitation of electron transport in the b(6)f complex may also arise from steric constraints in the entry/exit portal for passage of the plastoquinol and -quinone to/from its oxidation site proximal to the iron-sulfur cluster.

Figure 1. The electron transport chain of oxygenic photosynthesis

Formation of the trans-membrane Δμ̃_H+, by the linear electron transport chain, which extends from the H₂O oxidation site to that for reduction of NADP⁺, by H⁺ translocation in the PSII reaction center and cytochrome b₆f complex is shown, along with the utilization of this gradient by the ATP synthase. The rate-limitation in the linear ETC is associated with the oxidation of plastoquinol (PQH₂; see PQ in lipid bilayer) by the b₆f complex, which involves the structure-based entry/exit of the quinol-quinone to/from the [2Fe-2S] cluster of the iron-sulfur protein, and electron/proton transfer from the quinol to the cluster. Cyt b₆f (PDB ID 2E74), cytochrome b₆f; Fd (PDB ID 1EWY), ferredoxin; FNR (PDB ID 1EWY), ferredoxin-NADP⁺-reductase; PC (PDB ID 2Q5B), plastocyanin; PSII (PDB ID 3ARC) and PSI (PDB ID 1JB0), photosynthetic reaction center complexes.

Figure 2

(A) Crystal structure of dimeric cytochrome b₆f complex from the cyanobacterium, M. laminosus [(4) (PDB ID 2E74)], drawn in ribbon format, showing the eight subunits of the complex and five redox prosthetic groups, hemes f, b_p, b_n, the unique heme c_n (59, 60) and the [2Fe-2S] cluster present in each monomer. The two other prosthetic groups in each monomer, are a single chlorophyll a molecule ligated to two H₂O molecules (4) and, separated by a distance of 14 Å in each monomer, a single β-carotene molecule (61, 1, 2). Color code: cytochrome f TMH (orange), cytochrome b₆ (pink), Iron-Sulfur protein (yellow), subunit IV (cyan), small subunits with single TMH are PetG (wheat), PetL (red), PetM (gray) and PetN (green). (B) p-side quinol/quinone binding site and 11 × 12 Å p-side entry/exit portal (Q_p portal) for plastoquinol/plastoquinone; [2Fe-2S] cluster is shown as orange and yellow spheres, orange for Fe, S for sulfur; (C) Quinone/quinol binding site inferred from the crystal structure obtained in the presence of the p-side quinone-analogue inhibitor, tridecyl-stigmatellin (TDS, blue and red) (4) [n. b., at the high concentrations used for crystallization, the TDS was found to also bind in an n-side quinone-binding niche]. C and D helices belong to cyt b₆ (pink) and F and G- helices to subunit IV (cyan). (D) Plastoquinol substrate of cyt b₆f; number of isoprenoid groups in extended tail of the plastoquinol molecule, n=9.

Figure 2

(A) Crystal structure of dimeric cytochrome b₆f complex from the cyanobacterium, M. laminosus [(4) (PDB ID 2E74)], drawn in ribbon format, showing the eight subunits of the complex and five redox prosthetic groups, hemes f, b_p, b_n, the unique heme c_n (59, 60) and the [2Fe-2S] cluster present in each monomer. The two other prosthetic groups in each monomer, are a single chlorophyll a molecule ligated to two H₂O molecules (4) and, separated by a distance of 14 Å in each monomer, a single β-carotene molecule (61, 1, 2). Color code: cytochrome f TMH (orange), cytochrome b₆ (pink), Iron-Sulfur protein (yellow), subunit IV (cyan), small subunits with single TMH are PetG (wheat), PetL (red), PetM (gray) and PetN (green). (B) p-side quinol/quinone binding site and 11 × 12 Å p-side entry/exit portal (Q_p portal) for plastoquinol/plastoquinone; [2Fe-2S] cluster is shown as orange and yellow spheres, orange for Fe, S for sulfur; (C) Quinone/quinol binding site inferred from the crystal structure obtained in the presence of the p-side quinone-analogue inhibitor, tridecyl-stigmatellin (TDS, blue and red) (4) [n. b., at the high concentrations used for crystallization, the TDS was found to also bind in an n-side quinone-binding niche]. C and D helices belong to cyt b₆ (pink) and F and G- helices to subunit IV (cyan). (D) Plastoquinol substrate of cyt b₆f; number of isoprenoid groups in extended tail of the plastoquinol molecule, n=9.

Fig. 3

Motion of ISP soluble domain required for productive electron transfer in the cytochrome b₆f complex. Crystal structures of the cyt b₆f complex show the [2Fe-2S] cluster of the ISP soluble domain at a distance of 25.9 Å – 27.2 Å from heme f (Table 3) in a membrane proximal position. Electron transfer cannot proceed at a millisecond time scale over such a large distance. The ISP soluble domain, and possibly the luminal domain of cytochrome f as well, must undergo a motion to position the [2Fe-2S] cluster in proximity (16–18 Å) to heme f in a membrane distal position. Color scheme: Cyt f, light green; ISP, blue; Cyt b₆, subIV, Pet G, L, M, N, yellow; Hemes f, b_p, b_n, red; heme c_n, gray; [2Fe-2S] cluster, brown-yellow.

Fig. 4

Structures of cytochrome f with a displaced heme position. (A) The cyt f soluble domain (left) consists of a 75 Å long extended β sheet structure which is larger than the cyt c₁ soluble domain (right). In this restricted space, the cyt b₆f ISP soluble domain does not have the same conformational freedom as the cyt bc₁ ISP soluble domain. Hence, the ISP of cyt b₆f may undergo motion on a smaller scale as demonstrated by the relative insensitivity of the ISP hinge to mutations that restrict mobility (51). (B) For physiologically relevant electron transfer from the [2Fe-2S] cluster of the ISP soluble domain to heme f in cyt b₆f, the [2Fe-2S] cluster-heme f distance must be 16 Å – 18 Å. This may involve a small scale movement of the cyt f soluble domain as seen between the cyt b₆f crystal structures obtained from M. laminosus (PDB ID 2E74, black) and C. reinhardtii (PDB ID 1Q90, red). Structural superposition performed in PyMol between conserved residues of the cyt f trans-membrane helix (PDB ID 2E74, chain D, residues 258–277; PDB ID 1Q90, chain A, residues 255–274) using a C^α pair fitting algorithm (RMSD=0.84 Å). (C) In the superposed structure of cyt f from M. laminosus and C. reinhardtii, the heme f Fe atoms are separated by 3.8 Å. (M. l., Mastigocladus laminosus, C. r. Chlamydomonas reinhardtii).

Figure 5

The p-side quinone entry/exit portal. (A) Amino acid sequences of segment of F-helix of subunit IV of cytochrome b₆f complex showing conservation of proline residues at positions 105 and 112. (B) Schematic of trans-membrane F- and C-helices that define the p-side entry/exit portal for plastoquinol-plastoquinone transfer proximal to [2Fe-2S] cluster in the b₆f complex. Two proline residues, 105 and 112, cause a “bent” conformation of the wild-type trans-membrane F-helix of subunit IV relative to the trans-membrane C-helix of the cyt b subunit.

Figure 6

Anticipated effects on the time course of cytochrome f reduction, measured by the absorbance change at 556 nm, the alpha-band peak of the ‘reduced-oxidized’ difference spectrum vs. 540 nm as a reference of the different genetic constructs that are predicted to change the size of the PQH₂/PQ portal and thereby the rate-limiting step in the linear electron transport chain that can be assayed by the time course of cytochrome f reduction. (A) Introduction of additional proline residues designed to increase the portal size would increase the rate, i. e., decrease the half-time, of cyt f reduction, as seen in the trend from the blue → red → green → yellow traces that describe the time course of reduction after cytochrome f is oxidized by the red (μsec) light flash. (B) Change of Pro105 and Pro112 to Ala would cause a decrease in the rate of cyt f reduction (red, green traces) relative to the wild type (blue), with the effect predicted to be greater if both Pro→Ala changes are made (yellow trace).