Pathways for proton release during ubihydroquinone oxidation by the bc ₁ complex

Abstract

Quinol oxidation by the bc 1 complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the Q o site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c 1 . Effects of mutations to the ISP that change the redox potential and͞or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or ␤-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H ؉ transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.The bc 1 complex family of enzymes plays a central role in all the main pathways of energy conversion, and the photosynthetic apparatus of Rhodobacter sphaeroides exemplifies one of the simplest of these (1-5). This system is convenient experimentally because of the ease with which electron transfer can be initiated by illumination (6). X-ray crystallographic structures of mitochondrial complexes (7-10) contain at their core the three catalytic subunits, cytochrome (cyt) b, cyt c 1 , and the Rieske iron-sulfur protein (ISP), common to the bacterial enzymes (11-13). Homology models of these show that the catalytic superstructure is highly conserved, as had been expected from studies of the mechanism, which is essentially the same in the two systems (1-5). The bc 1 complex catalyzes the oxidation of quinol and the reduction of cyt c (or c 2 ) through a modified Q cycle (1-6, 14-17). Two separate internal electron transfer chains connect three catalytic sites. At one site, heme c 1 is oxidized by cyt c 2 . Two catalytic sites in cyt b are involved in oxidation or reduction of ubiquinone. In the bifurcated reaction at the quinol-oxidizing site (the Q o site), one electron from quinol is passed to the ISP, which transfers it to cyt c 1 , while the semiquinone produced is oxidized by another chain consisting of the two b hemes of cyt b. At the quinone-reducing site (Q i -site), electrons fr...

Mechanism of Ubiquinol Oxidation by the bc1 Complex: Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors

“…The structures suggest that this occurs through protonation of Glu-272 and rotation of the side chain, followed by dissociation and exit of the H + via the water chain proposed in ref 47 and discussed above ( Figure 7B,C) (49). Since rotation of Glu-272 away from the pocket is required to allow movement to the proximal domain, this would suggest that deprotonation of the semiquinone occurs before oxidation (after step c and before step d above), so that the semiquinone anion moves and donates to heme b L (steps d and e).…”

“…Earlier work from this lab had shown that the rate of quinol oxidation is determined by the probability of quinol binding, as shown by the secondorder nature of the reaction (15,21,50). We have more recently shown evidence that the oxidized ISP in its dissociated form reacts to form the complex and that the pH dependence of the rate in the acidic range reflects the availability of this species (41,42,49). Neither binding process contributes to the activation barrier, which is in the reactions after formation of the complex.…”

“…For E272, both these roles are indicated. Rotation to the out positions removes a physical blockage of the proximal domain, and a functional role in proton release seems likely (49). In the absence of evidence for a firmly bound quinone in the site, the inhibition of electron transfer by mutations in the proximal domain could most readily be explained by a need for movement of the semiquinone into this domain to bring it closer to cyt b L to allow rapid electron transfer.…”

“…As noted above, Glu-272 rotates to point into the binding pocket and form a H-bonds with the OH of stigmatellin. We believe that this rotation is of importance in release of the second proton from the site (49). Hydrophobic residues F275, F129, and L282 rotate to accommodate the inhibitor.…”

Ironies in photosynthetic electron transport: a personal perspective

“…In a 1984 paper on this subject (Widger et al 1984b), which I was proud to have sponsored by Warren Butler, we noted the conserved PEWY (proline-glutamic acid-tryptophan-tyrosine) sequence, in which the E has subsequently been implicated in the lumen-side pathway of proton transfer from ubiquinol in the bacterial Cyt bc 1 complex (Crofts et al 1999). Given the apparent split of the 8 transmembrane helix mitochondrial Cyt b to a 4 helix Cyt b 6 and a 3-helix subunit IV, the questions were: (i) why was the apparently highly conserved mitochondrial and photosynthetic bacterial Cyt b gene divided in approximately half in oxygenic photosynthesis, where Cyt b 6 is the N-terminal half, the four helix heme-binding homologue of the mitochondrial cytochrome; (ii) was a relatively large 8-helix protein split into 2 fragments, subunit IV and Cyt b 6 (Widger et al 1984b), or were Cyt b 6 and subunit IV fused to form the 8-subunit Cyt b of the Cyt bc 1 complex (Schütz et al 1994)?…”