Principle Features of the Structure

At a resolution of 3.0-3.1 A, the structure of the b6f complex is almost identical in the 2.5-3.0 x 109 year old thermophilic cyanobacterium, M. laminosus and the green alga, C. reinhardtii that appeared ~109 years later [6]. The hetero-oligomeric b6f complex consists of eight tightly bound subunits (Figure 7.1b), four large (MW = 17.5-32.3 kDa, cytb, cytf, subunit IV (suIV)),andfour small(MW = 3.30-4.06kDa, PetN, G, M. L) subunits, present in a 1 : 1 stoichiometry [7-9]. The dimeric complex contains 26 transmembrane helices, 13 in each monomer. The four large subunits, cyt f cyt b6, the Rieske [2Fe - 2S] protein, and subunit IV (red, blue, yellow, and purple in Figure 7.1b) bind the redox prosthetic groups of the complex, four hemes (1 bound by cytf, 3 by cyt b6), and one [2Fe-2S] cluster. Of the three hemes bound by the cytochrome b polypeptide, two are classical bis- histidine coordinated, non-covalently bound b-type hemes; the third is a novel heme, Cn, covalently bound (c-type) to Cys35 on the stromal n-side of the transmembrane helix A (TMH-A) of

the cyt b polypeptide (Figure 7.2a). The binding sites of PQ/PQH2 have been inferred from the binding sites of quinone analog inhibitors 28-10]. In addition, one chlorophyll a (green, Figure 7.1b) and one P-carotene (orange, Figure 7.1b) are present in each monomer 2 11-13] . The porphyrin ring of the Chl is inserted between the TMH . F and TMH . G, and the Chl phytyl chain is inserted through the portal in the "roof' of the inter-monomer quinone exchange cavity. The P-carotene is inserted through the petM and petG small subunits of the peripheral picket fence-like structure (green, Figure 7.1b) and extends to the E helix of sulV, a distance of closest approach of 14 A to the Chl a. The picket fence-like structure of the four small subunits is a unique aspect of the structure, not only with respect to the bc1 complex, but relative to all integral membrane proteins. It has been proposed that the picket fence and P-carotene toothpick may have an organizing function in assembly of the complex into the membrane [14].

7.1 Structure of the Cytochrome b6f Comples; Comparison with the Cytochrome bc1 Comples 157

Figure 7.1 (a) Electron transfer and proton pumping chain of oxygenic photosynthesis showing the pathway of electron transfer across the membrane from the luminal to the stromal side of Photosystem II reaction center (3.OA resolution; [3]) from water as the electron donor. Absorption of four photons and concomitant transfer of four electrons to the stromal n-side results in reduction of two plastoquinone (PQ) molecules, formation of one O2 molecule from two waters, that is, 2H2O ^ 02 + 4e- + 4 H+, and deposition of the 4 H+ on the lumen side of the membrane. The reduced plastoquinone (PQH2), which is hydrophobic, resides in a quinone "pool" (approximately 10-15 molecules per reaction center) in the center of the membrane bilayer, in which it can diffuse and enter the -5000A3 inter-monomer quinone exchange cavity. (b) Ribbon diagram of the native symmetric dimeric b6f complex from the thermophilic cyanobacterium, Mastigocladus laminosus, solved to a resolution of 3.0A [6], which contains 8 polypeptide subunits and 13 transmembrane helices, and whose electron density shows 7 prosthetic groups (four hemes, one [2Fe-2S] cluster, 1 chlorophyll a, and 1 P-carotene), and which has a dimer molecular weight of 217000. The "large" subunits are seen to bind the redox prosthetic groups (heme f, 2 hemes b, heme cn, the [2Fe-2S] cluster, and the single chlorophyll a and P-carotene. The function of the Chl a, whose porphyrin ring plane is inserted in a lipid-like manner between the F and G helices of subunit IV, whose 20 carbon phytyl tail is inserted into the quinone exchange cavity through the small lumenal p-side portal in the quinone exchange cavity, is not known. A function of the P-carotene is presumably to quench the Chl a excited triplet state, although the two molecules are seen in the structure to be separated by 14A, much too long a distance to allow quenching by wave function overlap. The four small subunits (petG, L, M, and N) are uniquely arranged in a picket fence fashion on the periphery of each monomer of the complex. (c) Once in the inter-monomer cavity, PQH2 (Em7 = +90mV) is oxidized to the neutral and anionic plasto-semiquinone, PQ^ and PQ'-, respectively, by the [2Fe-2S] cluster. (Em7 = +290 mV) of the Rieske iron-sulfur protein, depositing two H+ per electron transferred to the electrochemically positive (p) side of the membrane. The pathway of interheme b (bp -> bn) 1-electron transfer that results in stromal n- side reduction of plastoquinone to quinol (PQ -> PQH2), concomitant with uptake of two H+, is shown. From the binding of the quinone analogue inhibitor TDS or NQNO as a ligand to heme cn [9] in the position distal to heme bn, it was inferred that this site on the edge of the intermonomer quinone exchange cavity, is the physiological stromal n-side binding of the quinone, and thus the interface to the relatively mobile quinone in the cavity. The proximity (3.5 A) of heme bn to cn and their strong interaction is indicated by EPR analysis [17, 89], and binding of the quinone analog inhibitors to an axial position of heme cn. The presence of heme cn and the coupled hemes bn-cn may avoid formation of plasto-semiquinone that could be formed by heme bn reduction of O2, which could then generate ROS. PQ reduction may occur through 2-electron reduction by the coupled hemes bn-cn (Figure 7.4).

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