Cytochrome bc1-cy fusion complexes reveal the distance constraints for functional electron transfer between photosynthesis components.
ABSTRACT: Photosynthetic (Ps) growth of purple non-sulfur bacteria such as Rhodobacter capsulatus depends on the cyclic electron transfer (ET) between the ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductases (cyt bc1 complex), and the photochemical reaction centers (RC), mediated by either a membrane-bound (cyt c(y)) or a freely diffusible (cyt c2) electron carrier. Previously, we constructed a functional cyt bc1-c(y) fusion complex that supported Ps growth solely relying on membrane-confined ET ( Lee, D.-W., Ozturk, Y., Mamedova, A., Osyczka, A., Cooley, J. W., and Daldal, F. (2006) Biochim. Biophys. Acta 1757, 346-352 ). In this work, we further characterized this cyt bc1-c(y) fusion complex, and used its derivatives with shorter cyt c(y) linkers as "molecular rulers" to probe the distances separating the Ps components. Comparison of the physicochemical properties of both membrane-embedded and purified cyt bc1-c(y) fusion complexes established that these enzymes were matured and assembled properly. Light-activated, time-resolved kinetic spectroscopy analyses revealed that their variants with shorter cyt c(y) linkers exhibited fast, native-like ET rates to the RC via the cyt bc1. However, shortening the length of the cyt c(y) linker decreased drastically this electronic coupling between the cyt bc1-c(y) fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt c(y) linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc1-c(y) fusion complexes and the RC and their surrounding light harvesting proteins was very short. These findings support the notion that membrane-bound Ps components form large, active structural complexes that are "hardwired" for cyclic ET.
Project description:Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt c(y). In species like R. capsulatus, cyt c(y) functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c(y) (S-c(y)), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt c(y). This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c(y) regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c(y) variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c(y) was about 340 mV, similar to that of cyts c2 or c(y). Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-c(y) was distinct from those seen with the cyt c2 or cyt c(y). The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c(y) interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-c(y) revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt c(y), whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.
Project description:The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron?sulfur protein (ISP) with its Fe2S2 cluster, cyt c1 and cyt b, forming two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the electron transfer pathways originating from QH2 oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc1 and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Qo site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc1 activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc1. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH2 oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe2S2 cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH2 oxidation at the Qo site of cyt bc1.
Project description:Complex III or the cytochrome (cyt) bc1 complex constitutes an integral part of the respiratory chain of most aerobic organisms and of the photosynthetic apparatus of anoxygenic purple bacteria. The function of cyt bc1 is to couple the reaction of electron transfer from ubiquinol to cytochrome c to proton pumping across the membrane. Mechanistically, the electron transfer reaction requires docking of its Rieske iron-sulfur protein (ISP) subunit to the quinol oxidation site (QP) of the complex. Formation of an H-bond between the ISP and the bound substrate was proposed to mediate the docking. Here we show that the binding of oxazolidinedione-type inhibitors famoxadone, jg144, and fenamidone induces docking of the ISP to the QP site in the absence of the H-bond formation both in mitochondrial and bacterial cyt bc1 complexes, demonstrating that ISP docking is independent of the proposed direct ISP-inhibitor interaction. The binding of oxazolidinedione-type inhibitors to cyt bc1 of different species reveals a toxophore that appears to interact optimally with residues in the QP site. The effect of modifications or additions to the toxophore on the binding to cyt bc1 from different species could not be predicted from structure-based sequence alignments, as demonstrated by the altered binding mode of famoxadone to bacterial cyt bc1.
Project description:The cytochrome bc (cyt bc) complexes are involved in Q-cycling; they oxidize membrane quinols by high-potential electron acceptors, such as cytochromes or plastocyanin, and generate transmembrane proton gradient. In several prokaryotic lineages, and also in plant chloroplasts, the catalytic core of the cyt bc complexes is built of a four-helical cytochrome b (cyt b) that contains three hemes, a three-helical subunit IV, and an iron-sulfur Rieske protein (cytochrome b6 f-type complexes). In other prokaryotic lineages, and also in mitochondria, the cyt b subunit is fused with subunit IV, yielding a seven- or eight-helical cyt b with only two hemes (cyt bc1 -type complexes). Here we present an updated phylogenomic analysis of the cyt b subunits of cyt bc complexes. This analysis provides further support to our earlier suggestion that (1) the ancestral version of cyt bc complex contained a small four-helical cyt b with three hemes similar to the plant cytochrome b6 and (2) independent fusion events led to the formation of large cyts b in several lineages. In the search for a primordial function for the ancestral cyt bc complex, we address the intimate connection between the cyt bc complexes and photosynthesis. Indeed, the Q-cycle turnover in the cyt bc complexes demands high-potential electron acceptors. Before the Great Oxygenation Event, the biosphere had been highly reduced, so high-potential electron acceptors could only be generated upon light-driven charge separation. It appears that an ancestral cyt bc complex capable of Q-cycling has emerged in conjunction with the (bacterio)chlorophyll-based photosynthetic systems that continuously generated electron vacancies at the oxidized (bacterio)chlorophyll molecules.
Project description:Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome?bc1 complex (Complex?III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome?bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3?Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N-terminus of cyt.?c1 was found to greatly enhance the interaction between cyt.?b and cyt.?c1 , and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome?bc1 complex in an extreme thermal environment.
Project description:The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c1, which form two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the pathways of bifurcated electron transfers emanating from QH2 oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn2+ binding amino acid residues often correlate with proton uptake or release pathways. Earlier, using combined EXAFS and structural studies, we identified Zn coordinating residues of mitochondrial and bacterial cyt bc1. In this work, using the genetically tractable bacterial cyt bc1, we substituted each of the proposed Zn binding residues with non-protonatable side chains. Among these mutants, only the His291Leu substitution destroyed almost completely the Qo site catalysis without perturbing significantly the redox properties of the cofactors or the assembly of the complex. In this mutant, which is unable to support photosynthetic growth, the bifurcated electron transfer reactions that result from QH2 oxidation at the Qo site, as well as the associated proton(s) release, were dramatically impaired. Based on these findings, on the putative role of His291 in liganding Zn, and on its solvent exposed and highly conserved position, we propose that His291 of cyt b is critical for proton release associated to QH2 oxidation at the Qo site of cyt bc1.
Project description:The respiratory chain cytochrome bc1 complex (cyt bc1) is a major target of numerous antibiotics and fungicides. All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site. The primary cause of resistance to bc1 inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc1 to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc1. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc1 with IC50 values of 85.0 ?M and 69.2 ?M, respectively, indicating that it targets the essential subunits of cyt bc1 complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.
Project description:The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX- strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX- cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1beta. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.
Project description:Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll-protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.
Project description: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 bc(1) complex (and possibly the homologous b(6)f complex in chloroplasts) employs the [2Fe2S] cluster domain as a device to shuttle electrons from ubihydroquinone to cyt c(1) (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 Q(o) 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.