No abstract

In crystals of the key respiratory and photosynthetic electron transfer protein called ubihydroquinone:cytochrome (cyt) c oxidoreductase or cyt bc 1, the extrinsic [2Fe2S] cluster domain of its Fe-S subunit assumes several conformations, suggesting that it may move during catalysis. Herein, using Rhodobacter capsulatus mutants that have modifications in the hinge region of this subunit, we were able to reveal this motion kinetically. Thus, the bc1 complex (and possibly the homologous b6f complex in chloroplasts) employs the [2Fe2S] cluster domain as a device to shuttle electrons from ubihydroquinone to cyt c1 (or cyt f ). We demonstrate that this domain movement is essential for cyt bc 1 function, because a mutant enzyme with a nonmoving Fe-S subunit has no catalytic activity, and one with a slower movement has lower activity. This motion is apparently designed with a natural frequency slow enough to assure productive Qo site charge separation but fast enough not to be rate limiting. These findings add the unprecedented function of intracomplex electron shuttling to large-scale domain motions in proteins and may well provide a target for cyt bc 1 antibiotics.Rhodobacter capsulatus ͉ photosynthetic and respiratory electron transfer ͉ mitochondrial complex III ͉ protein domain motion ͉ Rieske Fe-S subunit W hen different crystal structures reveal dramatically different protein conformations, large amplitude domain movements are often inferred. However, in only a few cases such as myosin (1), flagellar motor (2), and ATP synthase (3, 4) have such movements been visualized. The cytochrome (cyt) bc 1 (or its cyt b 6 f counterpart in chloroplasts) is a key component of respiratory and photosynthetic electron transfer chains (5, 6). Recent crystal structures of the mitochondrial cyt bc 1 have revealed that the extrinsic [2Fe2S] cluster domain of the Fe-S subunit occupies various locations within this enzyme complex (7-10). It has been observed in either a position proximal to the ubihydroquinone (QH 2 ) oxidation catalytic site (Q o position) from which it takes electrons or a position close to cyt c 1 subunit (c 1 position) to which it donates electrons (Fig. 1). Because of the large distances observed between the electron-donating and electron-accepting cofactors of the cyt bc 1 in the different structures, no one of these locations can support sufficiently rapid electron tunneling (11) to meet the observed turnover rates (12, 13) and the specific substrate-product interactions (14) that occur at the QH 2 oxidation site. Thus, an unprecedented intracomplex electron shuttle motion to transfer electrons during catalysis has been suggested (8). However, neither the presumably essential movement nor the electron transfer associated with it has been visualized before this work.In light-activated energy transduction systems, such as the one provided by the photosynthetic bacterium R. capsulatus, a short flash of light (Ͻ10-s duration) can activate the photochemical reaction center, thereby inducing oxidation of two equival...

The ubihydroquinone:cytochrome (cyt) 1 c oxidoreductase, or bc 1 complex, is a key component of both respiratory and photosynthetic electron transfer chains (1-3). Using three redoxactive subunits (cyt b, cyt c 1 , and the iron-sulfur protein) and two active sites (Q o and ubiquinone reduction sites), it catalyzes the transfer of electrons from ubihydroquinone (QH 2 ) to a c-type cyt according to a mechanism known as the modified ubiquinone (Q) cycle (4, 5). This electron transfer is coupled to a proton transport across the membrane and contributes to the generation of the electrochemical gradient subsequently used for ATP synthesis via the ATP synthase. The key of the bc 1 complex energetics relies on the bifurcation of electrons at the Q o site. Upon QH 2 oxidation at this catalytic site one electron is transferred to a high potential chain constituted of a [2Fe-2S] cluster carried by the iron-sulfur subunit and a c-type cyt borne by the cyt c 1 subunit. The other electron is transferred to a low potential chain constituted of two b-type hemes (high potential b-type heme and low potential b-type heme (b L )) and then to a Q or a semiubiquinone radical at the ubiquinone reduction site, all carried by the cyt b subunit.Over the years, several hypotheses including the double occupancy model (6 -8), the proton-gated charge-transfer mechanism (9, 10), the formation of a stable intermediate between the semiubiquinone at the Q o site and the reduced iron-sulfur subunit until the second electron is transferred to heme b L (11), the rolling over of this semiubiquinone from a [2Fe-2S] proximal to a heme b L proximal position during QH 2 oxidation (12-14), and the redox exchange between the two monomers of a dimeric enzyme complex (15, 16) have been put forward to rationalize why the electrons emanating from QH 2 oxidation follow two thermodynamically different pathways. These models are based on kinetic data, analyses of the rate-limiting steps, energetics considerations, electron paramagnetic resonance (EPR) spectroscopy, and more recently crystallographic data. Indeed, during the past 4 years, several structures for mitochondrial bc 1 complexes obtained in the presence or absence of various inhibitors have been solved and revealed different conformations of the iron-sulfur subunit cluster domain in the enzyme complex (17-21). Similar data have also been obtained using electron microscopy with plastohydroquinone plastocyanin oxidoreductase, a homologue of the bc 1 complex in chloroplasts (22). These findings led collectively to the idea that the iron-sulfur subunit may move during QH 2 catalysis (17-19). More recently, this hypothesis has been confirmed by biochemical genetics (23-28) and kinetic analyses (23, 29) of various mutants located in the flexible region of the iron-sulfur subunit (hinge) linking its cluster domain and membrane anchor.Although we now know that oxidation of QH 2 at the Q o site is followed by an apparently concerted transfer of the two electrons, the movement of the iron-sulfur subunit, and the...

The ubihydroquinone:cytochrome c oxidoreductase, or bc(1) complex, functions according to a mechanism known as the modified Q cycle. Recent crystallographic data have revealed that the extrinsic domain containing the [2Fe2S] cluster of the Fe-S subunit of this enzyme occupies different positions in various crystal forms, suggesting that this subunit may move during ubihydroquinone oxidation. As in these structures the hydrophobic membrane anchor of the Fe-S subunit remains at the same position, the movement of the [2Fe2S] cluster domain would require conformational changes of the hinge region linking its membrane anchor to its extrinsic domain. To probe the role of the hinge region, Rhodobacter capsulatus bc(1) complex was used as a model, and various mutations altering the hinge region amino acid sequence, length, and flexibility were obtained. The effects of these modifications on the bc(1) complex function and assembly were investigated in detail. These studies demonstrated that the nature of the amino acid residues located in the hinge region (positions 43-49) of R. capsulatus Fe-S subunit was not essential per se for the function of the bc(1) complex. Mutants with a shorter hinge (up to five amino acid residues deletion) yielded functional bc(1) complexes, but contained substoichiometric amounts of the Fe-S subunit. Moreover, mutants with increased rigidity or flexibility of the hinge region altered both the function and the assembly or the steady-state stability of the bc(1) complex. In particular, the extrinsic domain of the Fe-S subunit of a mutant containing six proline residues in the hinge region was shown to be locked in a position similar to that seen in the presence of stigmatellin. Interestingly, the latter mutant readily overcomes this functional defect by accumulating an additional mutation which shortens the length of the hinge. These findings indicate that the hinge region of the Fe-S subunit of bacterial bc(1) complexes has a remarkable structural plasticity.