Crystallographic investigation of the ubiquinone binding site of respiratory Complex II and its inhibitors.

The quinone binding site (Q-site) of Mitochondrial Complex II (succinate-ubiquinone oxidoreductase) is the target for a number of inhibitors useful for elucidating the mechanism of the enzyme. Some of these have been developed as fungicides or pesticides, and species-specific Q-site inhibitors may be useful against human pathogens. We report structures of chicken Complex II with six different Q-site inhibitors bound, at resolutions 2.0-2.4 Å. These structures show the common interactions between the inhibitors and their binding site. In every case a carbonyl or hydroxyl oxygen of the inhibitor is H-bonded to Tyr58 in subunit SdhD and Trp173 in subunit SdhB. Two of the inhibitors H-bond Ser39 in subunit SdhC directly, while two others do so via a water molecule. There is a distinct cavity that accepts the 2-substituent of the carboxylate ring in flutolanil and related inhibitors. A hydrophobic "tail pocket" opens to receive a side-chain of intermediate-length inhibitors. Shorter inhibitors fit entirely within the main binding cleft, while the long hydrophobic side chains of ferulenol and atpenin A5 protrude out of the cleft into the bulk lipid region, as presumably does that of ubiquinone. Comparison of mitochondrial and Escherichia coli Complex II shows a rotation of the membrane-anchor subunits by 7° relative to the iron­sulfur protein. This rotation alters the geometry of the Q-site and the H-bonding pattern of SdhB:His216 and SdhD:Asp57. This conformational difference, rather than any active-site mutation, may be responsible for the different inhibitor sensitivity of the bacterial enzyme.

Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex.

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatisaa3-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O2 and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)-1 min-1 at 30 µM NO. The rate of NO consumption proved to be proportional to that of O2 consumption, with 2.65 ± 0.19 molecules of NO being consumed per O2 molecule by the mycobacterial bcc-aa3. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)-1 min-1 at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa3 supercomplexes against NO stress.

In-vitro, SDH5-dependent flavinylation of immobilized human respiratory complex II flavoprotein.

Mitochondrial Complex II (Succinate: ubiquinone oxidoreductase) has a covalently bound FAD cofactor in its largest subunit (SDHA), which accepts electrons from oxidation of succinate during catalysis. The mechanism of flavin attachment, and factors involved, have not been fully elucidated. The recent report of an assembly factor SDH5 (SDHAF2, SDHE) required for flavinylation (Hao et al., 2009 Science 325, 1139-1142) raises the prospect of achieving flavinylation in a completely defined system, which would facilitate elucidation of the precise role played by SDH5 and other factors. At this time that goal has not been achieved, and the actual function of SDH5 is still unknown. We have developed a procedure for in-vitro flavinylation of recombinant human apo-SDHA, immobilized on Ni-IMAC resin by a His tag, in a chemically defined medium. In this system flavinylation has a pH optimum of 6.5 and is completely dependent on added SDH5. The results suggest that FAD interacts noncovalently with SDHA in the absence of SDH5. This system will be useful in understanding the process of flavinylation of SDHA and the role of SDH5 in this process.

Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3.

Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2-a]pyridine derivatives, is believed to target the pathogen's homolog of the mitochondrial bc1 complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a "supercomplex" with a cytochrome aa3-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa3 supercomplex using a strain of M. smegmatis that has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding and provide structural insight into a third class of the bc complex superfamily.

Solution behavior and crystallization of cytochrome bc1 in the presence of amphipols.

Detergents classically are used to keep membrane proteins soluble in aqueous solutions, but they tend to destabilize them. This problem can be largely alleviated thanks to the use of amphipols (APols), small amphipathic polymers designed to substitute for detergents. APols adsorb at the surface of the transmembrane region of membrane proteins, keeping them water-soluble while stabilizing them bio-chemically. Membrane protein/APol complexes have proven, however, difficult to crystallize. In this study, the composition and solution properties of complexes formed between mitochondrial cytochrome bc1 and A8-35, the most extensively used APol to date, have been studied by means of size exclusion chromatography, sucrose gradient sedimentation, and small-angle neutron scattering. Stable, monodisperse preparations of bc1/A8-35 complexes can be obtained, which, depending on the medium, undergo either repulsive or attractive interactions. Under crystallization conditions, diffracting three-dimensional crystals of A8-35-stabilized cytochrome bc1 formed, but only in the concomitant presence of APol and detergent.

Unanswered questions about the structure of cytochrome bc1 complexes.

X-ray crystal structures of bc1 complexes obtained over the last 15 years have provided a firm structural basis for our understanding of the complex. For the most part there is good agreement between structures from different species, different crystal forms, and with different inhibitors bound. In this review we focus on some of the remaining unexplained differences, either between the structures themselves or the interpretations of the structural observations. These include the structural basis for the motion of the Rieske iron-sulfur protein in response to inhibitors, a possible conformational change involving tyrosine132 of cytochrome (cyt) b, the presence of cis-peptides at the beginnings of transmembrane helices C, E, and H, the structural insight into the function of the so-called "Core proteins", different modelings of the retained signal peptide, orientation of the low-potential heme b, and chirality of the Met ligand to heme c1. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Crystal structure of the yeast vacuolar ATPase heterotrimeric EGC(head) peripheral stalk complex.

Vacuolar ATPases (V-ATPases) are multisubunit rotary motor proton pumps that function to acidify subcellular organelles in all eukaryotic organisms. V-ATPase is regulated by a unique mechanism that involves reversible dissociation into V1-ATPase and V0 proton channel, a process that involves breaking of protein interactions mediated by subunit C, the cytoplasmic domain of subunit "a" and three "peripheral stalks," each made of a heterodimer of E and G subunits. Here, we present crystal structures of a yeast V-ATPase heterotrimeric complex composed of EG heterodimer and the head domain of subunit C (C(head)). The structures show EG heterodimer folded in a noncanonical coiled coil that is stabilized at its N-terminal ends by binding to C(head). The coiled coil is disrupted by a bulge of partially unfolded secondary structure in subunit G and we speculate that this unique feature in the eukaryotic V-ATPase peripheral stalk may play an important role in enzyme structure and regulation by reversible dissociation.

Computational discovery of picomolar Q(o) site inhibitors of cytochrome bc1 complex.

A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Q(o) site inhibitors of the cytochrome bc(1) complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (K(i) = 881.80 nM, porcine bc(1)), the most potent compound 4f displayed 20 507-fold improved binding affinity (K(i) = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (K(i) = 83.00 pM) bound to the chicken bc(1) at 2.70 Å resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques.

Engineering domain-swapped binding interfaces by mutually exclusive folding.

Domain swapping is a mechanism for forming protein dimers and oligomers with high specificity. It is distinct from other forms of oligomerization in that the binding interface is formed by reciprocal exchange of polypeptide segments. Swapping plays a physiological role in protein-protein recognition, and it can also potentially be exploited as a mechanism for controlled self-assembly. Here, we demonstrate that domain-swapped interfaces can be engineered by inserting one protein into a surface loop of another protein. The key to facilitating a domain swap is to destabilize the protein when it is monomeric but not when it is oligomeric. We achieve this condition by employing the "mutually exclusive folding" design to apply conformational stress to the monomeric state. Ubiquitin (Ub) is inserted into one of six surface loops of barnase (Bn). The 38-Å amino-to-carboxy-terminal distance of Ub stresses the Bn monomer, causing it to split at the point of insertion. The 2.2-Å X-ray structure of one insertion variant reveals that strain is relieved by intermolecular folding with an identically unfolded Bn domain, resulting in a domain-swapped polymer. All six constructs oligomerize, suggesting that inserting Ub into each surface loop of Bn results in a similar domain-swapping event. Binding affinity can be tuned by varying the length of the peptide linkers used to join the two proteins, which modulates the extent of stress. Engineered, swapped proteins have the potential to be used to fabricate "smart" biomaterials, or as binding modules from which to assemble heterologous, multi-subunit protein complexes.

Conformationally linked interaction in the cytochrome bc(1) complex between inhibitors of the Q(o) site and the Rieske iron-sulfur protein.

The modified Q cycle mechanism accounts for the proton and charge translocation stoichiometry of the bc(1) complex, and is now widely accepted. However the mechanism by which the requisite bifurcation of electron flow at the Q(o) site reaction is enforced is not clear. One of several proposals involves conformational gating of the docking of the Rieske ISP at the Q(o) site, controlled by the stage of the reaction cycle. Effects of different Q(o)-site inhibitors on the position of the ISP seen in crystals may reflect the same conformational mechanism, in which case understanding how different inhibitors control the position of the ISP may be a key to understanding the enforcement of bifurcation at the Q(o) site (Table 1). Here we examine the available structures of cytochrome bc(1) with different Q(o)-site inhibitors and different ISP positions to look for clues to this mechanism. The effect of ISP removal on binding affinity of the inhibitors stigmatellin and famoxadone suggest a "mutual stabilization" of inhibitor binding and ISP docking, however this thermodynamic observation sheds little light on the mechanism. The cd(1) helix of cytochrome b moves in such a way as to accommodate docking when inhibitors favoring docking are bound, but it is impossible with the current structures to say whether this movement of α-cd(1) is a cause or result of ISP docking. One component of the movement of the linker between E and F helices also correlates with the type of inhibitor and ISP position, and seems to be related to the H-bonding pattern of Y279 of cytochrome b. An H-bond from Y279 to the ISP, and its possible modulation by movement of F275 in the presence of famoxadone and related inhibitors, or its competition with an alternate H-bond to I269 of cytochrome b that may be destabilized by bound famoxadone, suggest other possible mechanisms. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex.

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc(1) complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q(i) site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q(o) site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A(3) in a fashion similar to the Q(o) site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q(i) and the Q(o) sites of the fungal cytochrome bc(1) complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc(1) complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q(o) and Q(i) sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc(1) complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q(i) and Q(o) sites. Moreover, crystal structure of chicken cytochrome bc(1) complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q(i) and Q(o) sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc(1) inhibitor that acts at both of the active sites of this enzyme.

X-Ray absorption studies of Zn2+ binding sites in bacterial, avian, and bovine cytochrome bc1 complexes.

Binding of Zn2+ has been shown previously to inhibit the ubiquinol cytochrome c oxidoreductase (cyt bc1 complex). X-ray diffraction data in Zn-treated crystals of the avian cyt bc1 complex identified two binding sites located close to the catalytic Qo site of the enzyme. One of them (Zn01) might interfere with the egress of protons from the Qo site to the aqueous phase. Using Zn K-edge x-ray absorption fine-structure spectroscopy, we report here on the local structure of Zn2+ bound stoichiometrically to noncrystallized cyt bc1 complexes. We performed a comparative x-ray absorption fine-structure spectroscopy study by examining avian, bovine, and bacterial enzymes. A large number of putative clusters, built by combining information from first-shell analysis and metalloprotein databases, were fitted to the experimental spectra by using ab initio simulations. This procedure led us to identify the binding clusters with high levels of confidence. In both the avian and bovine enzyme, a tetrahedral ligand cluster formed by two His, one Lys, and one carboxylic residue was found, and this ligand attribution fit the crystallographic Zn01 location of the avian enzyme. In the chicken enzyme, the ligands were the His121, His268, Lys270, and Asp253 residues, and in the homologous bovine enzyme they were the His121, His267, Lys269, and Asp254 residues. Zn2+ bound to the bacterial cyt bc1 complex exhibited quite different spectral features, consistent with a coordination number of 6. The best-fit octahedral cluster was formed by one His, two carboxylic acids, one Gln or Asn residue, and two water molecules. It was interesting that by aligning the crystallographic structures of the bacterial and avian enzymes, this group of residues was found located in the region homologous to that of the Zn01 site. This cluster included the His276, Asp278, Glu295, and Asn279 residues of the cyt b subunit. The conserved location of the Zn2+ binding sites at the entrance of the putative proton release pathways, and the presence of His residues point to a common mechanism of inhibition. As previously shown for the photosynthetic bacterial reaction center, zinc would compete with protons for binding to the His residues, thus impairing their function as proton donors/acceptors.

Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the "oxaloacetate-inhibited" state.

Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115-1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the "malate-like intermediate" found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108-1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1-9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.

3-nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon oxidation by complex II, forms a covalent adduct with a catalytic base arginine in the active site of the enzyme.

We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 A resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg(297) as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator of succinate dehydrogenase, forms a covalent adduct with the side chain of Arg(297). The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg(297) to be increased by 83 Da and to have the potential of losing 44 Da, consistent with decarboxylation, during fragmentation.

Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc1 complex: a new crystal structure reveals an altered intramolecular hydrogen-bonding pattern.

Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex. Structure-activity relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28 A resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cytochrome b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density, the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alphaA helix.

Crystallization of mitochondrial respiratory complex II from chicken heart: a membrane-protein complex diffracting to 2.0 A.

A procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory complex II. The crystals have the potential to diffract to at least 2.0 A with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane-protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.

X-Ray Structure of Rhodobacter Capsulatus Cytochrome bc (1): Comparison with its Mitochondrial and Chloroplast Counterparts.

Ubihydroquinone: cytochrome (cyt)c oxidoreductase, or cyt bc (1), is a widespread, membrane integral enzyme that plays a crucial role during photosynthesis and respiration. It is one of the major contributors of the electrochemical proton gradient, which is subsequently used for ATP synthesis. The simplest form of the cyt bc (1) is found in bacteria, and it contains only the three ubiquitously conserved catalytic subunits: the Fe-S protein, cyt b and cyt c (1). Here we present a preliminary X-ray structure of Rhodobacter capsulatus cyt bc (1) at 3.8 A and compare it to the available structures of its homologues from mitochondria and chloroplast. Using the bacterial enzyme structure, we highlight the structural similarities and differences that are found among the three catalytic subunits between the members of this family of enzymes. In addition, we discuss the locations of currently known critical mutations, and their implications in terms of the cyt bc (1) catalysis.

Observations concerning the quinol oxidation site of the cytochrome bc1 complex.

A direct hydrogen bond between ubiquinone/quinol bound at the QO site and a cluster-ligand histidine of the iron-sulfur protein (ISP) is described as a major determining factor explaining much experimental data on position of the ISP ectodomain, electron paramagnetic resonance (EPR) lineshape and midpoint potential of the iron-sulfur cluster, and the mechanism of the bifurcated electron transfer from ubiquinol to the high and low potential chains of the bc1 complex.