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Identification of ubiquinol binding motifs at the Qo-site of the cytochrome bc1 complex.

ABSTRACT: Enzymes of the bc1 complex family power the biosphere through their central role in respiration and photosynthesis. These enzymes couple the oxidation of quinol molecules by cytochrome c to the transfer of protons across the membrane, to generate a proton-motive force that drives ATP synthesis. Key for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol oxidation by the bc1 complex is well enough characterized to allow exploration at the atomistic level, but details are still highly controversial. The controversy stems from the uncertain binding motifs of quinol at the so-called Qo active site of the bc1 complex. Here we employ a combination of classical all atom molecular dynamics and quantum chemical calculations to reveal the binding modes of quinol at the Qo-site of the bc1 complex from Rhodobacter capsulatus. The calculations suggest a novel configuration of amino acid residues responsible for quinol binding and support a mechanism for proton-coupled electron transfer from quinol to iron-sulfur cluster through a bridging hydrogen bond from histidine that stabilizes the reaction complex.

SUBMITTER: Barragan AM

PROVIDER: S-EPMC4297238 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex.

Project description:1. Recent results suggest that the major flux is carried by a monomeric function, not by an intermonomer electron flow. 2. The bifurcated reaction at the Qo-site involves sequential partial processes, - a rate limiting first electron transfer generating a semiquinone (SQ) intermediate, and a rapid second electron transfer in which the SQ is oxidized by the low potential chain. 3. The rate constant for the first step in a strongly endergonic, proton-first-then-electron mechanism, is given by a Marcus-Brønsted treatment in which a rapid electron transfer is convoluted with a weak occupancy of the proton configuration needed for electron transfer. 4. A rapid second electron transfer pulls the overall reaction over. Mutation of Glu-295 of cyt b shows it to be a key player. 5. In more crippled mutants, electron transfer is severely inhibited and the bell-shaped pH dependence of wildtype is replaced by a dependence on a single pK at ~8.5 favoring electron transfer. Loss of a pK ~6.5 is explained by a change in the rate limiting step from the first to the second electron transfer; the pK ~8.5 may reflect dissociation of QH. 6. A rate constant (<10(3)s(-1)) for oxidation of SQ in the distal domain by heme bL has been determined, which precludes mechanisms for normal flux in which SQ is constrained there. 7. Glu-295 catalyzes proton exit through H(+) transfer from QH, and rotational displacement to deliver the H(+) to exit channel(s). This opens a volume into which Q(-) can move closer to the heme to speed electron transfer. 8. A kinetic model accounts well for the observations, but leaves open the question of gating mechanisms. For the first step we suggest a molecular "escapement"; for the second a molecular ballet choreographed through coulombic interactions. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

| S-EPMC3995752 | biostudies-literature

Binding dynamics at the quinone reduction (Qi) site influence the equilibrium interactions of the iron sulfur protein and hydroquinone oxidation (Qo) site of the cytochrome bc1 complex.

Project description:Multiple instances of low-potential electron-transport pathway inhibitors that affect the structure of the cytochrome (cyt) bc(1) complex to varying degrees, ranging from changes in hydroquinone (QH(2)) oxidation and cyt c(1) reduction kinetics to proteolytic accessibility of the hinge region of the iron-sulfur-containing subunit (Fe/S protein), have been reported. However, no instance has been documented of any ensuing change on the environment(s) of the [2Fe-2S] cluster. In this work, this issue was addressed in detail by taking advantage of the increased spectral and spatial resolution obtainable with orientation-dependent electron paramagnetic resonance (EPR) spectroscopic analysis of ordered membrane preparations. For the first time, perturbation of the low-potential electron-transport pathway by Q(i)-site inhibitors or various mutations was shown to change the EPR spectra of both the cyt b hemes and the [2Fe-2S] cluster of the Fe/S protein. In particular, two interlinked effects of Q(i)-site modifications on the Fe/S subunit, one changing the local environment of its [2Fe-2S] cluster and a second affecting the mobility of this subunit, are revealed. Remarkably, different inhibitors and mutations at or near the Q(i) site induce these two effects differently, indicating that the events occurring at the Q(i) site affect the global structure of the cyt bc(1). Furthermore, occupancy of discrete Q(i)-site subdomains differently impede the location of the Fe/S protein at the Q(o) site. These findings led us to propose that antimycin A and HQNO mimic the presence of QH(2) and Q at the Q(i) site, respectively. Implications of these findings in respect to the Q(o)-Q(i) sites communications and to multiple turnovers of the cyt bc(1) are discussed.

| S-EPMC1360200 | biostudies-literature

The cytochrome b Zn binding amino acid residue histidine 291 is essential for ubihydroquinone oxidation at the Qo site of bacterial cytochrome bc1.

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.

| S-EPMC5033722 | biostudies-literature

The rate-limiting step in the cytochrome bc1 complex (Ubiquinol-Cytochrome c Oxidoreductase) is not changed by inhibition of cytochrome b-dependent deprotonation: implications for the mechanism of ubiquinol oxidation at center P of the bc1 complex.

Project description:Quinol oxidation at center P of the cytochrome bc(1) complex involves bifurcated electron transfer to the Rieske iron-sulfur protein and cytochrome b. It is unknown whether both electrons are transferred from the same domain close to the Rieske protein, or if an unstable semiquinone anion intermediate diffuses rapidly to the vicinity of the b(L) heme. We have determined the pre-steady state rate and activation energy (E(a)) for quinol oxidation in purified yeast bc(1) complexes harboring either a Y185F mutation in the Rieske protein, which decreases the redox potential of the FeS cluster, or a E272Q cytochrome b mutation, which eliminates the proton acceptor in cytochrome b. The rate of the bifurcated reaction in the E272Q mutant (<10% of the wild type) was even lower than that of the Y185F enzyme ( approximately 20% of the wild type). However, the E272Q enzyme showed the same E(a) (61 kJ mol(-1)) with respect to the wild type (62 kJ mol(-1)), in contrast with the Y185F mutation, which increased E(a) to 73 kJ mol(-1). The rate and E(a) of the slow reaction of quinol with oxygen that are observed after cytochrome b is reduced were unaffected by the E272Q substitution, whereas the Y185F mutation modified only its rate. The Y185F/E272Q double mutation resulted in a synergistic decrease in the rate of quinol oxidation (0.7% of the wild type). These results are inconsistent with a sequential "movable semiquinone" mechanism but are consistent with a model in which both electrons are transferred simultaneously from the same domain in center P.

| S-EPMC2682884 | biostudies-literature

The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Qo site of bacterial cytochrome bc1.

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.

| S-EPMC6317905 | biostudies-literature

Saccharomyces cerevisiae-based mutational analysis of the bc1 complex Qo site residue 279 to study the trade-off between atovaquone resistance and function.

Project description:The bc1 complex is central to mitochondrial bioenergetics and the target of the antimalarial drug atovaquone that binds in the quinol oxidation (Qo) site of the complex. Structural analysis has shown that the Qo site residue Y279 (Y268 in Plasmodium falciparum) is key for atovaquone binding. Consequently, atovaquone resistance can be acquired by mutation of that residue. In addition to the probability of amino acid substitution, the level of atovaquone resistance and the loss of bc1 complex activity that are associated with the novel amino acid would restrict the nature of resistance-driven mutations occurring on atovaquone exposure in native parasite populations. Using the yeast model, we characterized the effect of all the amino acid replacements resulting from a single nucleotide substitution at codon 279: Y279C, Y279D, Y279F, Y279H, Y279N, and Y279S (Y279C, D, F, H, N, and S). Two residue changes that required a double nucleotide substitution, Y279A and W, were added to the series. We found that mutations Y279A, C, and S conferred high atovaquone resistance but decreased the catalytic activity. Y279F had wild-type enzymatic activity and sensitivity to atovaquone, while the other substitutions caused a dramatic respiratory defect. The results obtained with the yeast model were examined in regard to atomic structure and compared to the reported data on the evolution of acquired atovaquone resistance in P. falciparum.

| S-EPMC4468654 | biostudies-literature

Atomistic determinants of co-enzyme Q reduction at the Qi-site of the cytochrome bc1 complex.

Project description:The cytochrome (cyt) bc1 complex is an integral component of the respiratory electron transfer chain sustaining the energy needs of organisms ranging from humans to bacteria. Due to its ubiquitous role in the energy metabolism, both the oxidation and reduction of the enzyme's substrate co-enzyme Q has been studied vigorously. Here, this vast amount of data is reassessed after probing the substrate reduction steps at the Qi-site of the cyt bc1 complex of Rhodobacter capsulatus using atomistic molecular dynamics simulations. The simulations suggest that the Lys251 side chain could rotate into the Qi-site to facilitate binding of half-protonated semiquinone - a reaction intermediate that is potentially formed during substrate reduction. At this bent pose, the Lys251 forms a salt bridge with the Asp252, thus making direct proton transfer possible. In the neutral state, the lysine side chain stays close to the conserved binding location of cardiolipin (CL). This back-and-forth motion between the CL and Asp252 indicates that Lys251 functions as a proton shuttle controlled by pH-dependent negative feedback. The CL/K/D switching, which represents a refinement to the previously described CL/K pathway, fine-tunes the proton transfer process. Lastly, the simulation data was used to formulate a mechanism for reducing the substrate at the Qi-site.

| S-EPMC5035994 | biostudies-literature

Insights into cytochrome bc1 complex binding mode of antimalarial 2-hydroxy-1,4-naphthoquinones through molecular modelling.

Project description:BackgroundMalaria persists as a major public health problem. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding.ObjectivesThe aim of this work was to perform molecular modelling studies of 2-hydroxy-1,4-naphthoquinones analogues of atovaquone on the Qo site of P. falciparum cytochrome bc1 complex (Pfbc1) to suggest structural modifications that could improve their antimalarial activity.MethodsWe have built the homology model of the cytochrome b (CYB) and Rieske iron-sulfur protein (ISP) subunits from Pfbc1 and performed the molecular docking of 41 2-hydroxy-1,4-naphthoquinones with known in vitro antimalarial activity and predicted to act on this target.FindingsResults suggest that large hydrophobic R2 substituents may be important for filling the deep hydrophobic Qo site pocket. Moreover, our analysis indicates that the H-donor 2-hydroxyl group may not be crucial for efficient binding and inhibition of Pfbc1 by these atovaquone analogues. The C1 carbonyl group (H-acceptor) is more frequently involved in the important hydrogen bonding interaction with His152 of the Rieske ISP subunit.Main conclusionsAdditional interactions involving residues such as Ile258 and residues required for efficient catalysis (e.g., Glu261) could be explored in drug design to avoid development of drug resistance by the parasite.

| S-EPMC5354616 | biostudies-literature

Natural Compound-derived Cytochrome bc1 Complex Inhibitors as Antifungal Agents.

Project description:The high incidence of fungal pathogens has become a global issue for crop protection. A promising strategy to control fungal plant infections is based on the use of nature-inspired compounds. The cytochrome bc1 complex is an essential component of the cellular respiratory chain and is one of the most important fungicidal targets. Natural products have played a crucial role in the discovery of cytochrome bc1 inhibitors, as proven by the development of strobilurins, one of the most important classes of crop-protection agents, over the past two decades. In this review, we summarize advances in the exploration of natural product scaffolds for the design and development of new bc1 complex inhibitors. Particular emphasis is given to molecular modeling-based approaches and structure-activity relationship (SAR) studies performed to improve the stability and increase the potency of natural precursors. The collected results highlight the versatility of natural compounds and provide an insight into the potential development of nature-inspired derivatives as antifungal agents.

| S-EPMC7583968 | biostudies-literature

Crystal structure of the yeast cytochrome bc1 complex with its bound substrate cytochrome c.

Project description:Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to integral membrane proteins. Specific and transient complexes need to be formed between the redox partners to ensure fast turnover. In respiration, the mobile electron carrier cytochrome c shuttles electrons from the cytochrome bc1 complex to cytochrome c oxidase. Despite extensive studies of this fundamental step of energy metabolism, the structures of the respective electron transfer complexes were not known. Here we present the crystal structure of the complex between cytochrome c and the cytochrome bc1 complex from Saccharomyces cerevisiae. The complex was crystallized with the help of an antibody fragment, and its structure was determined at 2.97-A resolution. Cytochrome c is bound to subunit cytochrome c1 of the enzyme. The tight and specific interactions critical for electron transfer are mediated mainly by nonpolar forces. The close spatial arrangement of the c-type hemes unexpectedly suggests a direct and rapid heme-to-heme electron transfer at a calculated rate of up to 8.3 x 10(6) s(-1). Remarkably, cytochrome c binds to only one recognition site of the homodimeric multisubunit complex. Interestingly, the occupancy of quinone in the Qi site is higher in the monomer with bound cytochrome c, suggesting a coordinated binding and reduction of both electron-accepting substrates. Obviously, cytochrome c reduction by the cytochrome bc1 complex can be regulated in response to respiratory conditions.

| S-EPMC122428 | biostudies-literature

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