Cyt Bc1 Research Articles

Biological characterization of fenpicoxamid, a new fungicide with utility in cereals and other crops.

BACKGROUNDThe development of novel highly efficacious fungicides that lack cross‐resistance is extremely desirable. Fenpicoxamid (Inatreq™ active) possesses these characteristics and is a member of a novel picolinamide class of fungicides derived from the antifungal natural product UK‐2A.RESULTSFenpicoxamid strongly inhibited in vitro growth of several ascomycete fungi, including Zymoseptoria tritici (EC50, 0.051 mg L−1). Fenpicoxamid is converted by Z. tritici to UK‐2A, a 15‐fold stronger inhibitor of Z. tritici growth (EC50, 0.0033 mg L−1). Strong fungicidal activity of fenpicoxamid against driver cereal diseases was confirmed in greenhouse tests, where activity on Z. tritici and Puccinia triticina matched that of fluxapyroxad. Due to its novel target site (Qi site of the respiratory cyt bc1 complex) for the cereals market, fenpicoxamid is not cross‐resistant to Z. tritici isolates resistant to strobilurin and/or azole fungicides. Across multiple European field trials Z. tritici was strongly controlled (mean, 82%) by 100 g as ha−1 applications of fenpicoxamid, which demonstrated excellent residual activity.CONCLUSIONSThe novel chemistry and biochemical target site of fenpicoxamid as well as its lack of cross‐resistance and strong efficacy against Z. tritici and other pathogens highlight the importance of fenpicoxamid as a new tool for controlling plant pathogenic fungi. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Emergence of cytochrome bc complexes in the context of photosynthesis.

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 b 6 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 bc 1‐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 b 6 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.

Hydrogen Bonding to the Substrate Is Not Required for Rieske Iron-Sulfur Protein Docking to the Quinol Oxidation Site of Complex III

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.

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

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.

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

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.

Overall energy conversion efficiency of a photosynthetic vesicle.

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytb⁢c1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%-5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.

Atovaquone and ELQ-300 Combination Therapy as a Novel Dual-Site Cytochrome bc1 Inhibition Strategy for Malaria.

Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone (ATV) blocks the quinol oxidase (Qo) site of cyt bc1, while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Qi) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Qo site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc1 is a valuable strategy for antimalarial combination therapy and that Qi site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor ATV.

Chlorophyll a is the crucial redox sensor and transmembrane signal transmitter in the cytochrome b6f complex. Components and mechanisms of state transitions from the hydrophobic mismatch viewpoint

The cytochrome (cyt) b6f complex is involved in the transmembrane redox signaling that triggers state transitions in cyanobacteria and chloroplasts. However, the components and molecular mechanisms are still unclear. In an attempt to solve this long-standing problem, we first focused on the unknown role of a single chlorophyll a (Chla) in cyt b6f with a new approach based on Chla structural properties. Various b6f X-ray crystal structures were analyzed to identify their differences, which correlate with differences in Chla molecular volume. We found that the distance of the Rieske [2Fe-2S] cluster to Chla correlates with the distance between a pair of residues at the Qo-site and the distance between a pair of residues at the opposite membrane side. These correlations were accompanied by the rotation of a key peripheral residue and by changes in the hydrophobic thickness of cyt b6f. Parallel analysis of cyt bc1 crystal structures allowed us to conclude that Chla acts as the crucial redox sensor and transmembrane signal transmitter in b6f for changes in the plastoquinone pool redox state. The hydrophobic mismatch induced by the changed hydrophobic thickness of cyt b6f is the driving force for the structural reorganizations of the photosynthetic apparatus during induction and the progression of state transitions in cyanobacteria and chloroplasts. A mechanism for LHCII kinase activation in chloroplasts is also proposed. Our understanding of the dynamic structural changes in bc-complexes during turnover at the Qo-site and state transitions is augmented by the time-sequence ordering of 56 bc crystal structures.

Inhibition of cytochrome bc1 as a strategy for single-dose, multi-stage antimalarial therapy.

Single-dose therapies for malaria have been proposed as a way to reduce the cost and increase the effectiveness of antimalarial treatment. However, no compound to date has shown single-dose activity against both the blood-stage Plasmodium parasites that cause disease and the liver-stage parasites that initiate malaria infection. Here, we describe a subset of cytochrome bc1 (cyt bc1) inhibitors, including the novel 4(1H)-quinolone ELQ-400, with single-dose activity against liver, blood, and transmission-stage parasites in mouse models of malaria. Although cyt bc1 inhibitors are generally classified as slow-onset antimalarials, we found that a single dose of ELQ-400 rapidly induced stasis in blood-stage parasites, which was associated with a rapid reduction in parasitemia in vivo. ELQ-400 also exhibited a low propensity for drug resistance and was active against atovaquone-resistant P. falciparum strains with point mutations in cyt bc1. Ultimately, ELQ-400 shows that cyt bc1 inhibitors can function as single-dose, blood-stage antimalarials and is the first compound to provide combined treatment, prophylaxis, and transmission blocking activity for malaria after a single oral administration. This remarkable multi-stage efficacy suggests that metabolic therapies, including cyt bc1 inhibitors, may be valuable additions to the collection of single-dose antimalarials in current development.

Understanding the mechanism of atovaquone drug resistance in Plasmodium falciparum cytochrome b mutation Y268S using computational methods.

The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance.

Structural analysis of mitochondrial cytochrome bc1 complex with atovaquone bound reveals the molecular basis of antimalarial drug action

Results We determined the 3.0-A resolution X-ray structure of mitochondrial cyt bc1 from Saccharomyces cerevisiae with atovaquone bound in the catalytic Qo site [1]. The drug, which has a pKa of 6.9, forms a polarized H-bond between its ionized hydroxyl group and His181 of the Rieske protein subunit. Multiple non-polar interactions with side chains of cytochrome b residues stabilize hydroxynaphthoquinone and chlorophenyl-cyclohexyl groups. The cytochrome b sequence analysis showed that the majority of the interacting residues are conserved, so that atovaquone binding to the yeast cyt bc1 is likely to resemble the binding to the complexes of the target organisms. Conclusion The binding mode provides detailed insights in the molecular basis of broad target spectrum, species-specific efficacies and acquired resistances. This may aid drug development to control the spread of drug-resistant parasites.

The molecular evolution of the Qo motif.

Quinol oxidation in the catalytic quinol oxidation site (Qo site) of cytochrome (cyt) bc1 complexes is the key step of the Q cycle mechanism, which laid the ground for Mitchell’s chemiosmotic theory of energy conversion. Bifurcated electron transfer upon quinol oxidation enables proton uptake and release on opposite membrane sides, thus generating a proton gradient that fuels ATP synthesis in cellular respiration and photosynthesis. The Qo site architecture formed by cyt b and Rieske iron–sulfur protein (ISP) impedes harmful bypass reactions. Catalytic importance is assigned to four residues of cyt b formerly described as PEWY motif in the context of mitochondrial complexes, which we now denominate Qo motif as comprehensive evolutionary sequence analysis of cyt b shows substantial natural variance of the motif with phylogenetically specific patterns. In particular, the Qo motif is identified as PEWY in mitochondria, α- and ε-Proteobacteria, Aquificae, Chlorobi, Cyanobacteria, and chloroplasts. PDWY is present in Gram-positive bacteria, Deinococcus–Thermus and haloarchaea, and PVWY in β- and γ-Proteobacteria. PPWF only exists in Archaea. Distinct patterns for acidophilic organisms indicate environment-specific adaptations. Importantly, the presence of PDWY and PEWY is correlated with the redox potential of Rieske ISP and quinone species. We propose that during evolution from low to high potential electron-transfer systems in the emerging oxygenic atmosphere, cyt bc1 complexes with PEWY as Qo motif prevailed to efficiently use high potential ubiquinone as substrate, whereas cyt b with PDWY operate best with low potential Rieske ISP and menaquinone, with the latter being the likely composition of the ancestral cyt bc1 complex.

Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action.

Atovaquone, a substituted hydroxynaphthoquinone, is a potent antimalarial drug that acts by inhibiting the parasite's mitochondrial cytochrome bc1 complex (cyt bc1). Mutations in cyt bc1 confer atovaquone resistance. Here we describe the X-ray structure of mitochondrial cyt bc1 from Saccharomyces cerevisiae with atovaquone bound in the catalytic Qo site, at 3.0-Å resolution. A polarized H-bond to His181 of the Rieske protein in cyt bc1 traps the ionized hydroxyl group of the drug. Side chains of highly conserved cytochrome b residues establish multiple non-polar interactions with the napththoquinone group, whereas less-conserved residues are in contact with atovaquone's cyclohexyl-chlorophenyl tail. Our structural analysis reveals the molecular basis of atovaquone's broad target spectrum, species-specific efficacies and acquired resistances, and may aid drug development to control the spread of resistant parasites.

Studies on Inhibition of Respiratory Cytochrome bc1 Complex by the Fungicide Pyrimorph Suggest a Novel Inhibitory Mechanism

The respiratory chain cytochrome bc 1 complex (cyt bc 1) is a major target of numerous antibiotics and fungicides. All cyt bc 1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site. The primary cause of resistance to bc 1 inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc 1 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 bc 1. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc 1 with IC50 values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc 1 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.

Clarifying the Roles of Cardiolipin

Cardiolipins (CL) are uniquely structured double lipids that are universally found in membranes which couple electron transport and phosphorylation. The proposed roles of CL in these membranes include mainly two aspects: the effects on the structure and dynamics of the membranes' lipid component and the interplay with membrane-associated proteins. In previous work, we have studied both of these aspects by utilizing atomistic molecular dynamics simulations, starting with the general effects of CL on membrane properties and the interplay with ions [1], further moving on to the interactions with the membrane-embedded respiratory complex cyt bc1 [2]. The latter model system that is of greater interest comprises the entire cyt bc1 dimer of the purple photosynthetic bacterium Rhodobacter capsulatus embedded in a lipid bilayer, whose lipid composition mimics that of the inner mitochondrial membrane. Intriguingly CLs were observed to diffuse spontaneously to the dimer interface and to the immediate vicinity of the catalytic Qi-sites [2]. This observation is in agreement with experimental data, as CLs are indeed located close to the Qi-sites in several X-ray crystal structures of the complex. Importantly, our observations support the proposed role of CL in delivering protons for the non-reduced substrate forms in the active site.In ongoing work that we discuss here we focus more specifically on the roles of individual components of the proposed proton uptake pathway (CL, water, and individual protein residues) and on the atom-level reaction mechanism in the binding pocket. To this end, further MD simulations, QM calculations, and cite-directed mutagenesis experiments were employed. We also discuss whether there is a plausible pathway for substrate movement between the active sites through the lipid-filled insides of the complex, and the role of oxidative stress in cyt bc1 behavior.

Bridging a Gap Between Cytochrome Bc1 Complex Structure and Function

Cytochrome (cyt) bc1 complex has two active sites where energy-sustaining electron/proton transfer reactions occur. To determine the exact transfer steps involving the substrate quinone, the cyt bc1 complex of Rhodobacter capsulatus was studied using atomistic molecular dynamics simulations. The computations give clear-cut ideas how the proton transfer might take place at both of the active sites. The simulations strongly suggest that water-mediated interactions are needed to achieve quinol oxidation and to prevent short-circuit suppression at the Qo-site. In the case of the Qi-site, the simulations emphasize the role of negatively charged cardiolipin in quinone reduction. Together with site-directed mutagenesis this in silico approach provides long-awaited information regarding the structure-function relationship of the cyt bc1 complex.

Roles for Protons in Living Membranes: Cardiolipin

Cardiolipin (CL) in mitochondria has 2 unique headgroup conformations. In lipid bilayers (pK >7.0) it traps 2 protons forming patches. It also binds supercomplexes together where its phosphates bind cations. Patches trap protons with short, strong H-bonds (SSHBs) as described by Al Mildvan (Meth Enz 2007). CL's SSHBs trap protons its phosphates. They're tightly held due to insertion of CL's 4 chains into the bilayer. The H+, not available to water, has no affect the pH. Yet patches may act as a H+ antenna, pK >7.0, on both sides of the IM. X. Periole (JACS & Nature 2013) has identified CL-patch-binding-sites on the Xray structures of cyt bc1 & cyt oxidase, near the entrance to their putative H+ channels. Thus H+ delivery is on a microsecond (μs) timescale, like the chloroplast's chlorophyll antenna. The complexes pump on a msec timescale. CL patches are on the matrix side are near the F0 of the F0, F1 ATPase, the protons would charge F0 of the F0, F1, ATPase. SSHB formation minimally changes the pH on both membrane faces. Pumped protons have little ΔpH but a maximum ΔΨ. Junge & Oster have each shown that the F0 protons are driven by ΔΨ, not ΔpH.Living membrane surfaces are polyanionic, mostly on the lipid headgroups, tightly held together by their chains. Guoy-Chapman tells us that this decreases the surface pH. SHHB trapped protons have no effect on the pH. Finally, the ΔΨ of most living membranes is created by proton pumps on a msec timescale, but is adjusted by K-channels on a μs timescale. ΔΨ provokes wasteful leakage of H+s across the membrane. ATP energy pumps them back! Lipids, polyisoprenes, sterols, iso- and anteiso- chains, hopanoids and squalene inhibit this leakage.

Modulation of the redox state of quinones by light in Rhodobacter sphaeroides under anaerobic conditions

Illumination of intact cells of Rhodobacter sphaeroides under anaerobic conditions has a dual effect on the redox state of the quinone pool. A large oxidation of the quinone pool is observed during the first seconds following the illumination. This oxidation is suppressed by the addition of an uncoupler in agreement with a light-induced reverse electron transfer at the level of the complex I, present both in the non-invaginated part of the membrane and in the chromatophores. At longer dark times, this illumination increases the reducing power of the cells leading to a significant reduction of the others reaction centers (RCs). From the observation that a significant proportion of RCs could be reduced by the preillumination without affecting the numbers of charge separation for the RCs, we conclude that there is no rapid thermodynamic equilibrium between the quinones present in the non-invaginated part of the membrane and those localized in the chromatophores. Under anaerobic conditions where the chromatophores quinone pool is fully reduced, we deduce, on the basis of flash-induced fluorescence kinetics, that the reduced RCs are exclusively reoxidized by the quinone generated at the Q o site of the cyt bc 1 complex. The supramolecular association between a dimeric RC-LHI complex and one cyt bc 1 complex allows the confinement of a quinone between the RC-LHI directly associated to the cyt bc1 complex.

Parameterization of the prosthetic redox centers of the bacterial cytochrome bc 1 complex for atomistic molecular dynamics simulations

Cytochrome (cyt) bc 1 is a multi-subunit membrane protein complex that is a vital component of the respiratory and photosynthetic electron transfer chains both in bacteria and eukaryotes. Although the complex’s dimer structure has been solved using X-ray crystallography, it has not yet been studied in large-scale classical molecular dynamics (MD) simulations. In part, this is due to lack of suitable force field parameters, centered atomic point charges in particular, for the complex’s prosthetic redox centers. Accurate redox center charges are needed to depict realistically the inter-molecular interactions at different redox stages of the cyt bc 1 complex. Accordingly, here we present high-precision atomic point charges for the metal centers of the cyt bc 1 complex of Rhodobacter capsulatus derived from extensive density functional theory calculations, fitted using the restrained electrostatic potential methodology and combined with the CHARMM force field parameters. We also provide the Hartree–Fock charges for all substrate forms (quinol, quinone, and semiquinone) and the inhibitors antimycin and stigmatellin of the bacterial bc 1 complex. The accuracy of the parameterization scheme was verified by running a 200-ns MD simulation encompassing the entire cyt bc 1 complex embedded in a lipid bilayer and solvated with explicit water. The results indicate that these meticulously derived parameters are ready for running extensive MD simulations encompassing all biologically relevant stages of the cyt bc 1 complex reaction cycle.

Atomistic simulations indicate cardiolipin to have an integral role in the structure of the cytochrome bc1 complex

The reaction mechanism of the cytochrome (cyt) bc1 complex relies on proton and electron transfer to/from the substrate quinone/quinol, which in turn generate a proton gradient across the mitochondrial membrane. Cardiolipin (CL) have been suggested to play an important role in cyt bc1 function by both ensuring the structural integrity of the protein complex and also by taking part in the proton uptake. Yet, the atom-scale understanding of these highly charged four-tail lipids in the cyt bc1 function has remained quite unclear. We consider this issue through atomistic molecular dynamics simulations that are applied to the entire cyt bc1 dimer of the purple photosynthetic bacterium Rhodobacter capsulatus embedded in a lipid bilayer. We find CLs to spontaneously diffuse to the dimer interface to the immediate vicinity of the higher potential heme b groups of the complex's catalytic Qi-sites. This observation is in full agreement with crystallographic studies of the complex, and supports the view that CLs are key players in the proton uptake. The simulation results also allow us to present a refined picture for the dimer arrangement in the cyt bc1 complex, the novelty of our work being the description of the role of the surrounding lipid environment: in addition to the specific CL–protein interactions, we observe the protein domains on the positive side of the membrane to settle against the lipids. Altogether, the simulations discussed in this article provide novel views into the dynamics of cyt bc1 with lipids, complementing previous experimental findings.