Structures of Respiratory Supercomplex I+III2 Reveal Functional and Conformational Crosstalk.

The mitochondrial electron transport chain complexes are organized into supercomplexes (SCs) of defined stoichiometry, which have been proposed to regulate electron flux via substrate channeling. We demonstrate that CoQ trapping in the isolated SC I+III2 limits complex (C)I turnover, arguing against channeling. The SC structure, resolved at up to 3.8 Å in four distinct states, suggests that CoQ oxidation may be rate limiting because of unequal access of CoQ to the active sites of CIII2. CI shows a transition between "closed" and "open" conformations, accompanied by the striking rotation of a key transmembrane helix. Furthermore, the state of CI affects the conformational flexibility within CIII2, demonstrating crosstalk between the enzymes. CoQ was identified at only three of the four binding sites in CIII2, suggesting that interaction with CI disrupts CIII2 symmetry in a functionally relevant manner. Together, these observations indicate a more nuanced functional role for the SCs.

Incorporation of an Isohexide Subunit into the Endochin-like Quinolone Scaffold.

In order to improve the drug-likeness qualities, the antimalarial endochin-like quinolone (ELQ) scaffold has been modified by replacing the 4-(trifluoromethoxy)phenyl portion with an isoidide unit that is further adjustable by varying the distal O-substituents. As expected, the water solubilities of the new analogs are greatly improved, and the melting points are lower. However, the antimalarial potency of the new analogs is reduced to EC50 > 1 millimolar, a result ascribable to the hydrophilic nature of the new substitution.

The evolution of the human mitochondrial bc1 complex- adaptation for reduced rate of superoxide production?

The mitochondrial bc1 complex is a major source of mitochondrial superoxide. While bc1-generated superoxide plays a beneficial signaling role, excess production of superoxide lead to aging and degenerative diseases. The catalytic core of bc1 comprises three peptides -cytochrome b, Fe-S protein, and cytochrome c1. All three core peptides exhibit accelerated evolution in anthropoid primates. It has been suggested that the evolution of cytochrome b in anthropoids was driven by a pressure to reduce the production of superoxide. In humans, the bc1 core peptides exhibit anthropoid-specific substitutions that are clustered near functionally critical sites that may affect the production of superoxide. Here we compare the high-resolution structures of bovine, mouse, sheep and human bc1 to identify structural changes that are associated with human-specific substitutions. Several cytochrome b substitutions in humans alter its interactions with other subunits. Most significantly, there is a cluster of seven substitutions, in cytochrome b, the Fe-S protein, and cytochrome c1 that affect the interactions between these proteins at the tether arm of the Fe-S protein and may alter the rate of ubiquinone oxidation and the rate of superoxide production. Another cluster of substitutions near heme bH and the ubiquinone reduction site, Qi, may affect the rate of ubiquinone reduction and thus alter the rate of superoxide production. These results are compatible with the hypothesis that cytochrome b in humans (and other anthropoid primates) evolve to reduce the rate of production of superoxide thus enabling the exceptional longevity and exceptional cognitive ability of humans.

Generation of Electric Potential Difference by Chromatophores from Photosynthetic Bacteria in the Presence of Trehalose under Continuous Illumination.

Measurement of electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides associated with the surface of a nitrocellulose membrane filter (MF) impregnated with a phospholipid solution in decane or immersed into it in the presence of exogenous mediators and disaccharide trehalose demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The mediators were the ascorbate/N,N,N'N'-tetramethyl-p-phenylenediamine pair and ubiquinone-0 (electron donor and acceptor, respectively). Although stabilization of photoelectric responses upon long-term continuous illumination was observed for both variants of chromatophore immobilization, only the samples immersed into the MF retained the functional activity of reaction centers (RCs) for a month when stored in the dark at room temperature, which might be due to the preservation of integrity of chromatophore proteins inside the MF pores. The stabilizing effect of the bioprotector trehalose could be related to its effect on both the RC proteins and the phospholipid bilayer membrane. The results obtained will expand current ideas on the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into its electrochemical form.

Targeting the cytochrome bc1 complex for drug development in M. tuberculosis: review.

The terminal oxidases of the oxidative phosphorylation pathway play a significant role in the survival and growth of M. tuberculosis, targeting these components lead to inhibition of M. tuberculosis. Many drug candidates targeting various components of the electron transport chain in M. tuberculosis have recently been discovered. The cytochrome bc1-aa3 supercomplex is one of the most important components of the electron transport chain in M. tuberculosis, and it has emerged as the novel target for several promising candidates. There are two cryo-electron microscopy structures (PDB IDs: 6ADQ and 6HWH) of the cytochrome bc1-aa3 supercomplex that aid in the development of effective and potent inhibitors for M. tuberculosis. In recent years, a number of potential candidates targeting the QcrB subunit of the cytochrome bc1 complex have been developed. In this review, we describe the recently identified inhibitors that target the electron transport chain's terminal oxidase enzyme in M. tuberculosis, specifically the QcrB subunit of the cytochrome bc1 complex.

Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae.

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.

Decoding the Molecular Effects of Atovaquone Linked Resistant Mutations on Plasmodium falciparum Cytb-ISP Complex in the Phospholipid Bilayer Membrane.

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins' function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.

Design and synthesis of potent inhibitors of bc1 complex based on natural product neopeltolide.

Neopeltolide, a natural product isolated from deep-water sponge specimen of the family neopeltidae, has been proven to be a novel inhibitor of cytochrome bc1. In this study, a series of neopeltolide derivatives was designed by replacing the 14-membered macrolactone with indole ring and confirmed by 1H NMR, 13C NMR, and HRMS. Based on the binding mode of 12h with bc1 complex, the IC50 values of compounds 16a-f (ranging from 0.70 to 1.46 µM) were improved significantly than the ester derivatives 12a-u by replacing the ester with amide linker. Subsequently, the molecular docking results indicated that compound 16e could form a π-π interaction with Phe274 and two H-bonds with Glu271 and His161 and the latter H-bond was found to account for its high activity. The present work accelerates the discovery of novel bc1 complex inhibitors to deal with the resistance that the existing bc1 complex inhibitors are facing and provides a valuable idea for the design of new fungicides.

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

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.

A 3.3†Å-Resolution Structure of Hyperthermophilic Respiratory Complex†III Reveals the Mechanism of Its Thermal Stability.

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 Šresolution. 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.

Cbp3 and Cbp6 are dispensable for synthesis regulation of cytochrome b in yeast mitochondria.

Cytochrome b (Cytb) is the only mitochondrial encoded subunit from the bc1 complex. Cbp3 and Cbp6 are chaperones necessary for translation of the COB mRNA and Cytb hemylation. Here we demonstrate that their role in translation is dispensable in some laboratory strains, whereas their role in Cytb hemylation seems to be universally conserved. BY4742 yeast requires Cbp3 and Cbp6 for efficient COB mRNA translation, whereas the D273-10b strain synthesizes Cytb at wildtype levels in the absence of Cbp3 and Cbp6. Steady-state levels of Cytb are close to wildtype in mutant D273-10b cells, and Cytb forms non-functional, supercomplex-like species with cytochrome c oxidase, in which at least core 1, cytochrome c1, and Rieske iron-sulfur subunits are present. We demonstrated that Cbp3 interacts with the mitochondrial ribosome and with the COB mRNA in both BY4742 and D273-10b strains. The polymorphism(s) causing the differential function of Cbp3, Cbp6, and the assembly feedback regulation of Cytb synthesis is of nuclear origin rather than mitochondrial, and Smt1, a COB mRNA-binding protein, does not seem to be involved in the observed differential phenotype. Our results indicate that the essential role of Cbp3 and Cbp6 is to assist Cytb hemylation and demonstrate that in the absence of heme b, Cytb can form non-functional supercomplexes with cytochrome c oxidase. Our observations support that an additional protein or proteins are involved in Cytb synthesis in some yeast strains.

Cox15 interacts with the cytochrome bc1 dimer within respiratory supercomplexes as well as in the absence of cytochrome c oxidase.

The heme a molecule is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome c oxidase. Heme a is synthesized from heme o by a multi-spanning inner membrane protein, heme a synthase (Cox15 in the yeast Saccharomyces cerevisiae). The insertion of heme a is critical for cytochrome c oxidase function and assembly, but this process has not been fully elucidated. To improve our understanding of heme a insertion into cytochrome c oxidase, here we investigated the protein-protein interactions that involve Cox15 in S. cerevisiae In addition to observing Cox15 in homooligomeric complexes, we found that a portion of Cox15 also associates with the mitochondrial respiratory supercomplexes. When supercomplex formation was abolished, as in the case of stalled cytochrome bc1 or cytochrome c oxidase assembly, Cox15 maintained an interaction with select proteins from both respiratory complexes. In the case of stalled cytochrome bc1 assembly, Cox15 interacted with the late-assembling cytochrome c oxidase subunit, Cox13. When cytochrome c oxidase assembly was stalled, Cox15 unexpectedly maintained its interaction with the cytochrome bc1 protein, Cor1. Our results indicate that Cox15 and Cor1 continue to interact in the cytochrome bc1 dimer even in the absence of supercomplexes or when the supercomplexes are destabilized. These findings reveal that Cox15 not only associates with respiratory supercomplexes, but also interacts with the cytochrome bc1 dimer even in the absence of cytochrome c oxidase.

Mild phenotypes and proper supercomplex assembly in human cells carrying the homoplasmic m.15557G > A mutation in cytochrome b gene.

Respiratory complex III (CIII) is the first enzymatic bottleneck of the mitochondrial respiratory chain both in its native dimeric form and in supercomplexes. The mammalian CIII comprises 11 subunits among which cytochrome b is central in the catalytic core, where oxidation of ubiquinol occurs at the Qo site. The Qo- or PEWY-motif of cytochrome b is the most conserved through species. Importantly, the highly conserved glutamate at position 271 (Glu271) has never been studied in higher eukaryotes so far and its role in the Q-cycle remains debated. Here, we showed that the homoplasmic m.15557G > A/MT-CYB, which causes the p.Glu271Lys amino acid substitution predicted to dramatically affect CIII, induces a mild mitochondrial dysfunction in human transmitochondrial cybrids. Indeed, we found that the severity of such mutation is mitigated by the proper assembly of CIII into supercomplexes, which may favor an optimal substrate channeling and buffer superoxide production in vitro.

The cytochrome bc1 complex inhibitor Ametoctradin has an unusual binding mode.

Ametoctradin is an agricultural fungicide that selectively inhibits the cytochrome bc1 complex of oomycetes. Previous spectrophotometric studies using the purified cytochrome bc1 complex from Pythium sp. showed that Ametoctradin binds to the Qo-site of the enzyme. However, as modeling studies suggested a binding mode like that of the substrate ubiquinol, the possibility for a dual Qo- and Qi-site binding mode was left open. In this work, binding studies and enzyme assays with mitochondrial membrane preparations from Pythium sp. and an S. cerevisiae strain with a modified Qi-site were used to investigate further the binding mode of Ametoctradin. The results obtained argue that the compound could bind to both the Qo- and Qi-sites of the cytochrome bc1 complex and that its position or binding pose in the Qi-site differs from that of Cyazofamid and Amisulbrom, the two Qi-site-targeting, anti-oomycetes compounds. Furthermore, the data support the argument that Ametoctradin prefers binding to the reduced cytochrome bc1 complex. Thus, Ametoctradin has an unusual binding mode and further studies with this compound may offer the opportunity to better understand the catalytic cycle of the cytochrome bc1 complex.

Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle.

A key feature of the modified Q-cycle of the cytochrome bc1 and related complexes is a bifurcation of QH2 oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H+are released on complete turnover of the Q-cycle (2H+/ubiquinol (QH2) oxidized). We identified different partial processes of the Qo-site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron­sulfur protein. In the absence of Qo-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Qo-site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH2 on both turnovers. However, a minor lag in proton release found at pH 7 but not at pH 8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range.

Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V.

Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps-sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 µm column-enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-ß-D-maltoside. A total of 200 mg-300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes.

Design, synthesis and fungicidal evaluation of novel pyraclostrobin analogues.

A series of novel pyraclostrobin derivatives were designed and prepared as antifungal agents. Their antifungal activities were tested in vitro with five important phytopathogenic fungi, namely, Batrylis cinerea, Phytophthora capsici, Fusarium sulphureum, Gloeosporium pestis and Sclerotinia sclerotiorum using the mycelium growth inhibition method. Among these compounds, 5s displayed IC50 value of 0.57 µg/mL against Batrylis cinerea and 5k-II displayed IC50 value of 0.43 µg/mL against Sclerotinia sclerotiorum, which were close to that of the positive control pyraclostrobin (0.18 µg/mL and 0.15 µg/mL). Other compounds 5f, 5k-II, 5j, 5m and 5s also exhibited strong antifungal activity. Further enzymatic assay demonstrated compound 5s inhibited porcine bc1 complex with IC50 value of 0.95 µM. The statistical results from an integrated computational pipeline demonstrated the predicted total binding free energy for compound 5s is the highest. Consequently, compound 5s with the biphenyl-4-methoxyl side chain could serve as a new motif as inhibitors of bc1 complex and deserve to be further investigated.

The obligate respiratory supercomplex from Actinobacteria.

Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (Em(ISP)=+160mV, Em(bL)=-291mV, Em(bH)=-163mV, Em(cc)=+100mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (Em(CuA)=+150mV, Em(a/a3)=+143/+317mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity.

From low- to high-potential bioenergetic chains: Thermodynamic constraints of Q-cycle function.

The electrochemical parameters of all cofactors in the supercomplex formed by the Rieske/cytb complex and the SoxM/A-type O2-reductase from the menaquinone-containing Firmicute Geobacillus stearothermophilus were determined by spectroelectrochemistry and EPR redox titrations. All redox midpoint potentials (Em) were found to be lower than those of ubi- or plastoquinone-containing systems by a value comparable to the redox potential difference between the respective quinones. In particular, Em values of +200mV, -360mV, -220mV and -50mV (at pH7) were obtained for the Rieske cluster, heme bL, heme bH and heme ci, respectively. Comparable values of -330mV, -200mV and +120mV for hemes bL, bH and the Rieske cluster were determined for an anaerobic Firmicute, Heliobacterium modesticaldum. Thermodynamic constraints, optimization of proton motive force build-up and the necessity of ROS-avoidance imposed by the rise in atmospheric O2 2.5billionyears ago are discussed as putative evolutionary driving forces resulting in the observed redox upshift. The close conservation of the entire redox landscape between low and high potential systems suggests that operation of the Q-cycle requires the precise electrochemical tuning of enzyme cofactors to the quinone substrate as stipulated in P. Mitchell's hypothesis.

Internal switches modulating electron tunneling currents in respiratory complex III.

In different X-ray crystal structures of bc1 complex, some of the key residues of electron tunneling pathways are observed in different conformations; here we examine their relative importance in modulating electron transfer and propose their possible gating function in the Q-cycle. The study includes inter-monomeric electron transfer; here we provide atomistic details of the reaction, and discuss the possible roles of inter-monomeric electronic communication in bc(1) complex. Binding of natural ligands or inhibitors leads to local conformational changes which propagate through protein and control the conformation of key residues involved in the electron tunneling pathways. Aromatic-aromatic interactions are highly utilized in the communication network since the key residues are aromatic in nature. The calculations show that there is a substantial change of the electron transfer rates between different redox pairs depending on the different conformations acquired by the key residues of the complex.