Mechanism of enhanced superoxide production in the cytochrome b(6)f complex of oxygenic photosynthesis

Abstract

The specific rate of superoxide (O2(•-)) production in the purified active crystallizable cytochrome b6f complex, normalized to the rate of electron transport, has been found to be more than an order of magnitude greater than that measured in isolated yeast respiratory bc1 complex. The biochemical and structural basis for the enhanced production of O2(•-) in the cytochrome b6f complex compared to that in the bc1 complex is discussed. The higher rate of superoxide production in the b6f complex could be a consequence of an increased residence time of plastosemiquinone/plastoquinol in its binding niche near the Rieske protein iron-sulfur cluster, resulting from (i) occlusion of the quinone portal by the phytyl chain of the unique bound chlorophyll, (ii) an altered environment of the proton-accepting glutamate believed to be a proton acceptor from semiquinone, or (iii) a more negative redox potential of the heme bp on the electrochemically positive side of the complex. The enhanced rate of superoxide production in the b6f complex is physiologically significant as the chloroplast-generated reactive oxygen species (ROS) functions in the regulation of excess excitation energy, is a source of oxidative damage inflicted during photosynthetic reactions, and is a major source of ROS in plant cells. Altered levels of ROS production are believed to convey redox signaling from the organelle to the cytosol and nucleus.

Figure 1

The dimeric cytochrome b₆f complex (PDB ID 4H44). Prosthetic groups in the cytochrome b₆f complex. Hemes b_p (red), b_n (red) and c_n (black) are redox-active prosthetic groups located within the trans-membrane domain, and constitute the low-potential chain. Heme f (black) and [2Fe-2S] cluster (orange/yellow spheres) form the high-potential chain, and are associated with the p-side extrinsic domains of cytochrome f and ISP, respectively. A chlorophyll-a (green) and a β-carotene (yellow) are also associated with the complex. Polypeptides are shown as ribbons. Color code: cytochrome b₆ (cyt b₆), wheat; subunit IV (subIV), orange; cytochrome f (cyt f), cyan; iron-sulfur protein (ISP), yellow; PetL, red; PetM, green; PetG, blue; PetN, gray.

Figure 2

Trans-membrane electron transfer and Q-cycle mechanism in a schematic of the cytochrome b₆f complex, indicating the branching of electrons from the anionic semiquinone reductant, PQ_p∸ and heme b_p to O₂, the latter reactions responsible for formation of superoxide, O₂∸.

Figure 3

Representative original data traces for determination of rates of (A, upper traces) superoxide generation and (B, lower traces) electron transfer in isolated bc₁ and b₆f complexes. The electron transfer and superoxide generation activities were measured as described in Materials and Methods using cytochrome c as the electron acceptor for the yeast cyt bc₁, cyt b₆f from M. laminosus, and cyanobacterial plastocyanin for the b₆f complex from spinach. Traces averaged from two to four measurements are shown. The background fluorescence change measured for the reaction mixture before the addition of the enzyme was subtracted from the superoxide generation activity data (B, lower traces). The arrow indicates addition of 5 nM and 45 nM bc₁ complex, respectively, for reactions in the absence and presence of inhibitor, and 5 nM b₆f complex.

Figure 4

Role of conserved Glu (Pro-Glu-Trp-Tyr) in semiquinone deprotonation in cyochrome bc complexes. (A) In the bc₁ complex, the Glu271 residue (green/red stcks, labeled in blue) of the PEWY sequence is found to occupy two distinct locations-quinone proximal (PDB ID 3CX5) and heme b_p-proximal (PDB ID 1NTZ). In the heme b_p-proximal position, Glu271 inteacts with Tyr131 (cytochrome b polypeptide). The figure was generated by superposition of cytochrome bc₁ crystal structures (PDB IDs 1NTZ and 3CX5). The STG molecule and the quinone/STG-proximal Glu271 orientation were obtained from PDB ID 3CX5. (B) In the cytochrome b₆f complex crystal structure (PDB ID 4H44), Glu78 (green/red sticks, labeled in blue) of subunitIV, homologous to Glu271 in the bc₁ complex, is located in the heme b_p-proximal orientation. In this position, Glu78 interacts with Arg87 of cytochrome b₆ (b₆f), through a distance of 2.8 Å. Arg87 (b₆f) is replaced by Ala84 (cyan sticks) in the bc₁ complex. Arg83 of cytochrome b₆ (b₆f) and Arg80 of cytochrome b (bc₁) are conserved in their location. The quinone analog inhibitor TDS has been inserted into the figure from PDB ID 4H13 to mark the Q_p-site. The transmembrane helices (a-g) are labeled. The polypeptides are shown as ribbons. (C) Multiple sequence alignment of the cytochrome b₆ subunit of the cytochrome b₆f complex from prokaryotic cyanobacteria (Synechocystis PCC 6803, Synechococcus elongates PCC 6301, Nostoc PCC 7120, M. laminosus), a eukaryotic alga (C. reinhardtii), and higher plants (Arabidopsis thaliana and Spinacea oleracea). Arg83, Arg87 (trans-membrane helix B) and Tyr136 (trans-membrane helix C) are conserved in cytochrome b₆ polypeptide.