Conservation of lipid functions in cytochrome bc complexes

Abstract

Lipid binding sites and properties are compared in two sub-families of hetero-oligomeric membrane protein complexes known to have similar functions in order to gain further understanding of the role of lipid in the function, dynamics, and assembly of these complexes. Using the crystal structure information for both complexes, we compared the lipid binding properties of the cytochrome b(6)f and bc(1) complexes that function in photosynthetic and respiratory membrane energy transduction. Comparison of lipid and detergent binding sites in the b(6)f complex with those in bc(1) shows significant conservation of lipid positions. Seven lipid binding sites in the cyanobacterial b(6)f complex overlap three natural sites in the Chlamydomonas reinhardtii algal complex and four sites in the yeast mitochondrial bc(1) complex. The specific identity of lipids is different in b(6)f and bc(1) complexes: b(6)f contains sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol, whereas cardiolipin, phosphatidylethanolamine, and phosphatidic acid are present in the yeast bc(1) complex. The lipidic chlorophyll a and β-carotene (β-car) in cyanobacterial b(6)f, as well as eicosane in C. reinhardtii, are unique to the b(6)f complex. Inferences of lipid binding sites and functions were supported by sequence, interatomic distance, and B-factor information on interacting lipid groups and coordinating amino acid residues. The lipid functions inferred in the b(6)f complex are as follows: (i) substitution of a transmembrane helix by a lipid and chlorin ring, (ii) lipid and β-car connection of peripheral and core domains, (iii) stabilization of the iron-sulfur protein transmembrane helix, (iv) n-side charge and polarity compensation, and (v) β-car-mediated super-complex with the photosystem I complex.

Fig 1. Subunit organization and lipid binding sites in the cytochrome b₆f complex

(A) Dimeric cytochrome b₆f complex from Mastigocladus laminosus (PDB ID 2E74), showing the positions of the eight protein subunits. Side view, in plane of membrane. Color code: Cytochrome f/Pet A (yellow), cytochrome b₆/Pet B (cyan), Rieske Fe₂S₂ protein/Pet C (orange), subunit IV/Pet D (pink), Pet G (teal), Pet L (light brown), Pet M (green) and Pet N (gray). (B) Side view of M. laminosus cyt b₆f complex showing lipids, detergents and pigments. (C) Top view along the membrane normal of M. laminosus cytochrome b₆f complex (PDB ID 2E74) showing 26 trans-membrane helices and the 2-fold symmetry axis between the monomers. Lipids (MGDG₁ and MGDG₂, magenta and red) and a pigment (eicosane, blue) from the C. reinhardtii b₆f complex were superimposed on the M. laminosus b₆f structure by combining PDB ID 2E74 and PDB ID 1Q90. The TMH of cytochrome b₆ (A–D) and subunit IV (E–G), Rieske Iron-Sulfur protein, cytochrome f, and the peripheral Pet subunits are shown as cylinders. n, p, electrochemically negative and positive sides of the complex. (D) Neutral and anionic lipids in spinach cyt b₆f complex. Major lipids of spinach b₆f complex detected by liquid chromatography with mass spectrometry (89). (a). Positive ion mass spectrum of ammoniated neutral lipids (M+NH₄)⁺ after a reverse-phase separation of a chloroform extract of cytochrome b₆f complex. The 764.4 and 792.6 Da species are assigned as the (M+NH₄)⁺ ions of monogalactosyldiacylglycerol (MGDG) with 16:3, 18:3 and 18:3, 18:3 fatty acids respectively (C₄₃H₇₀O₁₀ and C₄₅H₇₄O₁₀ calculated mono-isotopic masses 746.49 & 774.53 Da, for the neutral species). The 926.7 and 954.6 Da species are assigned as the (M+NH₄)⁺ ions of digalactosyldiacylglycerol (DGDG) with 16:3, 18:3 and 18:3, 18:3 fatty acids respectively (C₄₉H₈₀O₁₅ and C₅₁H₈₄O₁₅ calculated mono-isotopic masses 908.55 & 936.58 Da, for the neutral species). (b). The negative-ion mass spectrum (m/z 700 – 870) of the same sample is shown. The 741.6 Da species is assigned as the (M-H⁺)⁻ ion of phosphatidylglycerol (PG) with 16:1 and 18:3 fatty acids (C₄₀H₇₁O₁₀P₁; calculated mono-isotopic mass 742.48 Da, for the neutral species). The ions at 815.4 and 837.6 Da are assigned as (M-H⁺)⁻ ions of sulfoquinovosyldiacylglycerol (SDG) with either 16:0, 18:3 or 18:3, 18:3 fatty acids respectively (C₄₃H₇₈O₁₂S₁ and C₄₅H₇₆O₁₂S₁ calculated mono-isotopic masses 816.51 & 838.49 Da, for the neutral species).

Fig 1. Subunit organization and lipid binding sites in the cytochrome b₆f complex

(A) Dimeric cytochrome b₆f complex from Mastigocladus laminosus (PDB ID 2E74), showing the positions of the eight protein subunits. Side view, in plane of membrane. Color code: Cytochrome f/Pet A (yellow), cytochrome b₆/Pet B (cyan), Rieske Fe₂S₂ protein/Pet C (orange), subunit IV/Pet D (pink), Pet G (teal), Pet L (light brown), Pet M (green) and Pet N (gray). (B) Side view of M. laminosus cyt b₆f complex showing lipids, detergents and pigments. (C) Top view along the membrane normal of M. laminosus cytochrome b₆f complex (PDB ID 2E74) showing 26 trans-membrane helices and the 2-fold symmetry axis between the monomers. Lipids (MGDG₁ and MGDG₂, magenta and red) and a pigment (eicosane, blue) from the C. reinhardtii b₆f complex were superimposed on the M. laminosus b₆f structure by combining PDB ID 2E74 and PDB ID 1Q90. The TMH of cytochrome b₆ (A–D) and subunit IV (E–G), Rieske Iron-Sulfur protein, cytochrome f, and the peripheral Pet subunits are shown as cylinders. n, p, electrochemically negative and positive sides of the complex. (D) Neutral and anionic lipids in spinach cyt b₆f complex. Major lipids of spinach b₆f complex detected by liquid chromatography with mass spectrometry (89). (a). Positive ion mass spectrum of ammoniated neutral lipids (M+NH₄)⁺ after a reverse-phase separation of a chloroform extract of cytochrome b₆f complex. The 764.4 and 792.6 Da species are assigned as the (M+NH₄)⁺ ions of monogalactosyldiacylglycerol (MGDG) with 16:3, 18:3 and 18:3, 18:3 fatty acids respectively (C₄₃H₇₀O₁₀ and C₄₅H₇₄O₁₀ calculated mono-isotopic masses 746.49 & 774.53 Da, for the neutral species). The 926.7 and 954.6 Da species are assigned as the (M+NH₄)⁺ ions of digalactosyldiacylglycerol (DGDG) with 16:3, 18:3 and 18:3, 18:3 fatty acids respectively (C₄₉H₈₀O₁₅ and C₅₁H₈₄O₁₅ calculated mono-isotopic masses 908.55 & 936.58 Da, for the neutral species). (b). The negative-ion mass spectrum (m/z 700 – 870) of the same sample is shown. The 741.6 Da species is assigned as the (M-H⁺)⁻ ion of phosphatidylglycerol (PG) with 16:1 and 18:3 fatty acids (C₄₀H₇₁O₁₀P₁; calculated mono-isotopic mass 742.48 Da, for the neutral species). The ions at 815.4 and 837.6 Da are assigned as (M-H⁺)⁻ ions of sulfoquinovosyldiacylglycerol (SDG) with either 16:0, 18:3 or 18:3, 18:3 fatty acids respectively (C₄₃H₇₈O₁₂S₁ and C₄₅H₇₆O₁₂S₁ calculated mono-isotopic masses 816.51 & 838.49 Da, for the neutral species).

Fig 1. Subunit organization and lipid binding sites in the cytochrome b₆f complex

(A) Dimeric cytochrome b₆f complex from Mastigocladus laminosus (PDB ID 2E74), showing the positions of the eight protein subunits. Side view, in plane of membrane. Color code: Cytochrome f/Pet A (yellow), cytochrome b₆/Pet B (cyan), Rieske Fe₂S₂ protein/Pet C (orange), subunit IV/Pet D (pink), Pet G (teal), Pet L (light brown), Pet M (green) and Pet N (gray). (B) Side view of M. laminosus cyt b₆f complex showing lipids, detergents and pigments. (C) Top view along the membrane normal of M. laminosus cytochrome b₆f complex (PDB ID 2E74) showing 26 trans-membrane helices and the 2-fold symmetry axis between the monomers. Lipids (MGDG₁ and MGDG₂, magenta and red) and a pigment (eicosane, blue) from the C. reinhardtii b₆f complex were superimposed on the M. laminosus b₆f structure by combining PDB ID 2E74 and PDB ID 1Q90. The TMH of cytochrome b₆ (A–D) and subunit IV (E–G), Rieske Iron-Sulfur protein, cytochrome f, and the peripheral Pet subunits are shown as cylinders. n, p, electrochemically negative and positive sides of the complex. (D) Neutral and anionic lipids in spinach cyt b₆f complex. Major lipids of spinach b₆f complex detected by liquid chromatography with mass spectrometry (89). (a). Positive ion mass spectrum of ammoniated neutral lipids (M+NH₄)⁺ after a reverse-phase separation of a chloroform extract of cytochrome b₆f complex. The 764.4 and 792.6 Da species are assigned as the (M+NH₄)⁺ ions of monogalactosyldiacylglycerol (MGDG) with 16:3, 18:3 and 18:3, 18:3 fatty acids respectively (C₄₃H₇₀O₁₀ and C₄₅H₇₄O₁₀ calculated mono-isotopic masses 746.49 & 774.53 Da, for the neutral species). The 926.7 and 954.6 Da species are assigned as the (M+NH₄)⁺ ions of digalactosyldiacylglycerol (DGDG) with 16:3, 18:3 and 18:3, 18:3 fatty acids respectively (C₄₉H₈₀O₁₅ and C₅₁H₈₄O₁₅ calculated mono-isotopic masses 908.55 & 936.58 Da, for the neutral species). (b). The negative-ion mass spectrum (m/z 700 – 870) of the same sample is shown. The 741.6 Da species is assigned as the (M-H⁺)⁻ ion of phosphatidylglycerol (PG) with 16:1 and 18:3 fatty acids (C₄₀H₇₁O₁₀P₁; calculated mono-isotopic mass 742.48 Da, for the neutral species). The ions at 815.4 and 837.6 Da are assigned as (M-H⁺)⁻ ions of sulfoquinovosyldiacylglycerol (SDG) with either 16:0, 18:3 or 18:3, 18:3 fatty acids respectively (C₄₃H₇₈O₁₂S₁ and C₄₅H₇₆O₁₂S₁ calculated mono-isotopic masses 816.51 & 838.49 Da, for the neutral species).

Fig 1. Subunit organization and lipid binding sites in the cytochrome b₆f complex

(A) Dimeric cytochrome b₆f complex from Mastigocladus laminosus (PDB ID 2E74), showing the positions of the eight protein subunits. Side view, in plane of membrane. Color code: Cytochrome f/Pet A (yellow), cytochrome b₆/Pet B (cyan), Rieske Fe₂S₂ protein/Pet C (orange), subunit IV/Pet D (pink), Pet G (teal), Pet L (light brown), Pet M (green) and Pet N (gray). (B) Side view of M. laminosus cyt b₆f complex showing lipids, detergents and pigments. (C) Top view along the membrane normal of M. laminosus cytochrome b₆f complex (PDB ID 2E74) showing 26 trans-membrane helices and the 2-fold symmetry axis between the monomers. Lipids (MGDG₁ and MGDG₂, magenta and red) and a pigment (eicosane, blue) from the C. reinhardtii b₆f complex were superimposed on the M. laminosus b₆f structure by combining PDB ID 2E74 and PDB ID 1Q90. The TMH of cytochrome b₆ (A–D) and subunit IV (E–G), Rieske Iron-Sulfur protein, cytochrome f, and the peripheral Pet subunits are shown as cylinders. n, p, electrochemically negative and positive sides of the complex. (D) Neutral and anionic lipids in spinach cyt b₆f complex. Major lipids of spinach b₆f complex detected by liquid chromatography with mass spectrometry (89). (a). Positive ion mass spectrum of ammoniated neutral lipids (M+NH₄)⁺ after a reverse-phase separation of a chloroform extract of cytochrome b₆f complex. The 764.4 and 792.6 Da species are assigned as the (M+NH₄)⁺ ions of monogalactosyldiacylglycerol (MGDG) with 16:3, 18:3 and 18:3, 18:3 fatty acids respectively (C₄₃H₇₀O₁₀ and C₄₅H₇₄O₁₀ calculated mono-isotopic masses 746.49 & 774.53 Da, for the neutral species). The 926.7 and 954.6 Da species are assigned as the (M+NH₄)⁺ ions of digalactosyldiacylglycerol (DGDG) with 16:3, 18:3 and 18:3, 18:3 fatty acids respectively (C₄₉H₈₀O₁₅ and C₅₁H₈₄O₁₅ calculated mono-isotopic masses 908.55 & 936.58 Da, for the neutral species). (b). The negative-ion mass spectrum (m/z 700 – 870) of the same sample is shown. The 741.6 Da species is assigned as the (M-H⁺)⁻ ion of phosphatidylglycerol (PG) with 16:1 and 18:3 fatty acids (C₄₀H₇₁O₁₀P₁; calculated mono-isotopic mass 742.48 Da, for the neutral species). The ions at 815.4 and 837.6 Da are assigned as (M-H⁺)⁻ ions of sulfoquinovosyldiacylglycerol (SDG) with either 16:0, 18:3 or 18:3, 18:3 fatty acids respectively (C₄₃H₇₈O₁₂S₁ and C₄₅H₇₆O₁₂S₁ calculated mono-isotopic masses 816.51 & 838.49 Da, for the neutral species).

Fig. 2. Substitution of a TMH in bc1 by a lipid and lipid-like chlorophyll a in the b6f complex

C-terminal trans-membrane 8^th helix (H-helix, orange) of cyt b subunit of yeast bc₁ complex (PDB ID 3CX5) interacts with the F- and G-helices. In superimposed b₆f (PDB ID 2E74) and bc₁ (PDB ID 3CX5) complexes, a DOPC lipid (yellow acyl tails, orange and red head group), together with the Chl a (green), protrudes from the space between the F- and G- helices (pink) and fills the niche occupied by the H TMH in the bc₁ complex. Protein chains of bc₁ subunits not shown.

Fig. 3. Lipid-mediated connection of peripheral monotopic Pet G, L, M, and N subunits and the polytopic core of the b₆f complex

The natural galactolipid MGDG₁ (green) fills the interstitial space between small Pet G, L, M, and N subunits of the C. reinhardtii cyt b₆f complex (PDB ID 1Q90). The artificial lipid DOPC_p (stick format, orange) occupies one of the niches between the small Pet subunits in the cyanobacterial cyt b₆f complex (PDB ID 2E74). Neighboring β – carotene shown in yellow. A second galactolipid, MGDG₂, is found inserted between the TMH of Pet G and the G-helix of sub IV of the algal cyt b₆f.

Fig 4. Sub-domain structure of cyt b₆f

The cyt b₆f monomer (PDB ID 2E74) is organized into two distinct sub-domains- (i) a polytopic sub-domain (shown in pink) that consists of cyt b₆ and subIV and constitutes the central portion of the dimer, and (ii) a single TMH sub-domain (shown in blue) that is composed of Pet L, M, N cyt f and ISP. This sub-domain is more peripheral in position. The interface between the two sub-domains is formed by the β–carotene molecule (yellow) and Pet G (teal). View of the cyt b₆f complex along the normal to the membrane (2-fold symmetry axis is shown at the inter-monomer interface).

Fig. 5. Acyl chain stabilization of the trans-membrane helix (TMH) of the Rieske iron-sulfur protein (ISP)

The acyl tails of a phosphatidic acid, PA (yellow, orange) of cyt bc₁ is wrapped around the ISP TMH. A UDM (white and red) molecule is located in a similar position in the cyanobacterial b₆f complex, while the niche is occupied by eicosane (green) in the algal b₆f complex. The cyanobacterial (PDB ID 2E74) and algal (PDB ID 1Q90) b₆f complex are superposed with the the yeast bc₁ (PDB ID 3CX5).

Fig. 6. n-side charge compensation

Conserved position for binding acidic lipid(s) (46) on the n-side of cyanobacterial b₆f (PDB ID 2E74) and bc₁ complexes (PDB ID 3CX5). (A) Three lipid binding sites (1 sulfolipid and 2 UDM molecules) are observed on the n-side of the inter-monomer cavity, close to the lipid-aqueous interface, in each monomer of the cyanobacterial b₆f complex (M. laminosus PDB ID 2E74). The head-group of the sulfolipid (color, wheat) is within an interaction distance (3.5 Å) of 1 UDM molecule (white and red), which interacts with another UDM molecule (pink and red, separation 3.8 Å). Disordered acyl chains of sulfolipid and detergent molecules can define a pathway for plastoquinone/plastoquinol exchange between the inter-monomer cavity and bilayer (2Fo-Fc electron density map contoured at σ=1.0 (~0.1 electrons/ų). The acidic sulfolipid molecule of cyt b₆f interacts with Lys275 (cyt f) and Arg16 and Asn20 (ISP). (B) The sulfolipid (wheat, transparent) is located close to the acidic PA (green) and detergent UDM (teal) of the bc₁ complex suggesting an important role for this lipid binding niche.

Fig. 6. n-side charge compensation

Conserved position for binding acidic lipid(s) (46) on the n-side of cyanobacterial b₆f (PDB ID 2E74) and bc₁ complexes (PDB ID 3CX5). (A) Three lipid binding sites (1 sulfolipid and 2 UDM molecules) are observed on the n-side of the inter-monomer cavity, close to the lipid-aqueous interface, in each monomer of the cyanobacterial b₆f complex (M. laminosus PDB ID 2E74). The head-group of the sulfolipid (color, wheat) is within an interaction distance (3.5 Å) of 1 UDM molecule (white and red), which interacts with another UDM molecule (pink and red, separation 3.8 Å). Disordered acyl chains of sulfolipid and detergent molecules can define a pathway for plastoquinone/plastoquinol exchange between the inter-monomer cavity and bilayer (2Fo-Fc electron density map contoured at σ=1.0 (~0.1 electrons/ų). The acidic sulfolipid molecule of cyt b₆f interacts with Lys275 (cyt f) and Arg16 and Asn20 (ISP). (B) The sulfolipid (wheat, transparent) is located close to the acidic PA (green) and detergent UDM (teal) of the bc₁ complex suggesting an important role for this lipid binding niche.

Fig 7. Super-complex formation; bridge to photosystem I

Cardiolipin of cyt bc₁ (green and red) is involved in stabilizing super-complexes with cyt c oxidase (55). Superposition of the bc₁ complex (PDB ID 3CX5) with the cyanobacterial b₆f complex (PDB ID 2E74) results in close spatial location of cardiolipin (acyl chains, green; orange and red are head group phosphorous and oxygen atoms respectively) and β–carotene (yellow), which has been suggested to mediate a cyt b₆f-PSI supercomplex (5).

Fig 8. n-side H⁺ antenna function

An acidic cardiolipin molecule (cyan), shared between the two monomers of the bc₁ complex (PDB ID 3CX5). Two detergent molecules (white/red and pink/red, one from each monomer) are found in the same position in the cyanobacterial cyt b₆f complex (M. laminosus, PDB ID 2E74) when the structures are superposed and may mark the position of a lipid molecule.