Complex II phosphorylation is triggered by unbalanced redox homeostasis in cells lacking complex III

A marked stimulation of complex II enzymatic activity was detected in cybrids bearing a homoplasmic MTCYB microdeletion causing disruption of both the activity and the assembly of complex III, but not in cybrids harbouring another MTCYB mutation affecting only the complex III activity. Moreover, complex II stimulation was associated with SDHA subunit tyrosine phosphorylation. Despite the lack of detectable hydrogen peroxide production, up-regulation of the levels of mitochondrial antioxidant defenses revealed a significant redox unbalance. This effect was also supported by the finding that treatment with N-acetylcysteine dampened the complex II stimulation, SDHA subunit tyrosine phosphorylation, and levels of antioxidant enzymes. In the absence of complex III, the cellular amount of succinate, but not fumarate, was markedly increased, indicating that enhanced activity of complex II is hampered due to the blockage of respiratory electron flow. Thus, we propose that complex II phosphorylation and stimulation of its activity represent a molecular mechanism triggered by perturbation of mitochondrial redox homeostasis due to severe dysfunction of respiratory complexes. Depending on the site and nature of the damage, complex II stimulation can either bypass the energetic deficit as an efficient compensatory mechanism, or be ineffectual, leaving cells to rely on glycolysis for survival.     

Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery.     

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.     

Design,synthesis, and evaluation of novel N-1 fluoroquinolone derivatives: Probing for binding contact with the active site tyrosine of gyrase

Structural studies of topoisomerase-fluoroquinolone-DNA ternary complexes revealed a cavity between the quinolone N-1 position and the active site tyrosine. Fluoroquinolone derivatives having positively charged or aromatic moieties extended from the N-1 position were designed to probe for binding contacts with the phosphotyrosine residue in ternary complex. While alkylamine, alkylphthalimide, and alkylphenyl groups introduced at the N-1 position afforded derivatives that maintained modest inhibition of the supercoiling activity of DNA gyrase, none retained ability to poison DNA gyrase. Thus, the addition of a large and/or long moiety at the N-1 position disrupts ternary complex formation, and retained ability to inhibit supercoiling is likely through interference with the strand breakage reaction. Two derivatives were found to possess inhibitory effects on the decatenation activity of human topoisomerase II.     

Spectroscopic and magnetochemical studies on the active site copper complex in galactose oxidase

Galactose oxidase is a radical copper oxidase, an enzyme making use of a covalently modified tyrosine residue as a free radical redox cofactor in alcohol oxidation catalysis. We report here a combination of spectroscopic and magnetochemical studies developing insight into the interactions between the active site Cu(II) and two distinct tyrosine ligands in the biological complex. One of the tyrosine ligands (Y495) is coordinated to the Cu(II) metal center as a phenolate in the resting enzyme and serves as a general base to abstract a proton from the coordinated substrate, thus activating it for oxidation. The structure of the resting enzyme is temperature-dependent as a consequence of an internal proton equilibrium associated with this tyrosine that mimics this catalytic proton transfer step. The other tyrosine ligand (Y272) is covalently crosslinked to a cysteine residue forming a tyrosine–cysteine dimer free radical redox site that is required for hydrogen atom abstraction from the activated substrate alkoxide. The presence of the free radical in the oxidized active enzyme results in formation of an EPR-silent Cu(II) complex shown by multifield magnetic saturation experiments to be a diamagnetic singlet arising from antiferromagnetic exchange coupling between the metal and radical spins. A paramagnetic contribution observed at higher temperature may be associated with thermal population of the triplet state, thus permitting an estimate of the magnitude of the isotropic exchange coupling (J>200 cm⁻¹, JS₁·S₂) in this complex. Structural correlations and the possible mechanistic significance of metal–radical coupling in the active enzyme are discussed.     

Binding Modes of Phthalocyanines to Amyloid β Peptide and Their Effects on Amyloid Fibril Formation

The inherent tendency of proteins to convert from their native states into amyloid aggregates is associated with a range of human disorders, including Alzheimer’s and Parkinson’s diseases. In that sense, the use of small molecules as probes for the structural and toxic mechanism related to amyloid aggregation has become an active area of research. Compared with other compounds, the structural and molecular basis behind the inhibitory interaction of phthalocyanine tetrasulfonate (PcTS) with proteins such as αS and tau has been well established, contributing to a better understanding of the amyloid aggregation process in these proteins. We present here the structural characterization of the binding of PcTS and its Cu(II) and Zn(II)-loaded forms to the amyloid β-peptide (Aβ) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aβ₄₀ revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aβ₄₀-PcTS complex, ascribed to a binding mode in which the planarity and hydrophobicity of the aromatic ring system in the phthalocyanine act as main structural determinants for the interaction. Our results demonstrated that formation of the Aβ₄₀-PcTS complex does not interfere with the progression of the peptide toward the formation of amyloid fibrils. On the other hand, conjugation of Zn(II) but not Cu(II) at the center of the PcTS macrocyclic ring modified substantially the binding profile of this phthalocyanine to Aβ₄₀ and became crucial to reverse the effects of metal-free PcTS on the fibril assembly of the peptide. Overall, our results provide a firm basis to understand the structural rules directing phthalocyanine-protein interactions and their implications on the amyloid fibril assembly of the target proteins; in particular, our results contradict the hypothesis that PcTS might have similar mechanisms of action in slowing the formation of a variety of pathological aggregates.     

The insert within the catalytic domain of tripeptidyl-peptidase II is important for the formation of the active complex.

Tripeptidyl-peptidase II (TPP II) is a large (Mr>10(6)) tripeptide-releasing enzyme with an active site of the subtilisin-type. Compared with other subtilases, TPP II has a 200 amino-acid insertion between the catalytic Asp44 and His264 residues, and is active as an oligomeric complex. This study demonstrates that the insert is important for the formation of the active high-molecular mass complex. A recombinant human TPP II and a murine TPP II were found to display different complex-forming characteristics when over-expressed in human 293-cells; the human enzyme was mainly in a nonassociated, inactive state whereas the murine enzyme formed active oligomers. This was surprising because native human TPP II is purified from erythrocytes as an active oligomeric complex, and the amino-acid sequences of the human and murine enzymes were 96% identical. Using a combination of chimeras and a single point mutant, the amino acid responsible for this difference was identified as Arg252 in the recombinant human sequence, which corresponds to a glycine in the murine sequence. As Gly252 is conserved in all sequenced variants of TPP II, the recombinant enzyme with Arg252 is atypical. Nevertheless, as Arg252 evidently interferes with complex formation, and this residue is close to the catalytic His264, it may also explain why oligomerization influences enzyme activity. The exact mechanism for how the G252R substitution interferes with complex formation remains to be determined, but will be of importance for the understanding of the unique properties of TPP II.     

Crystal structure of a type II dehydroquinate dehydratase-like protein from Bifidobacterium longum

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. Here we identify a Bifidobacterium longum protein with high sequence homology to type II DHQDs but no detectable DHQD activity under standard assay conditions. A crystal structure reveals that the B. longum protein adopts a DHQD-like tertiary structure but a distinct quaternary state. Apparently forming a dimer, the B. longum protein lacks the active site aspartic acid contributed from a neighboring protomer in the type II DHQD dodecamer. Relating to the absence of protein–protein interactions established in the type II DHQD dodecameric assembly, substantial conformational changes distinguish the would-be active site of the B. longum protein. As B. longum possess no other genes with homology to known DHQDs, these findings imply a unique DHQD activity within B. longum.     

Expression of Yeast NDI1 Rescues a Drosophila Complex I Assembly Defect

Defects in mitochondrial electron transport chain (ETC) function have been implicated in a number of neurodegenerative disorders, cancer, and aging. Mitochondrial complex I (NADH dehydrogenase) is the largest and most complicated enzyme of the ETC with 45 subunits originating from two separate genomes. The biogenesis of complex I is an intricate process that requires multiple steps, subassemblies, and assembly factors. Here, we report the generation and characterization of a Drosophila model of complex I assembly factor deficiency. We show that CG7598 (dCIA30), the Drosophila homolog of human complex I assembly factor Ndufaf1, is necessary for proper complex I assembly. Reduced expression of dCIA30 results in the loss of the complex I holoenzyme band in blue-native polyacrylamide gel electrophoresis and loss of NADH:ubiquinone oxidoreductase activity in isolated mitochondria. The complex I assembly defect, caused by mutation or RNAi of dCIA30, has repercussions both during development and adulthood in Drosophila, including developmental arrest at the pupal stage and reduced stress resistance during adulthood. Expression of the single-subunit yeast alternative NADH dehydrogenase, Ndi1, can partially or wholly rescue phenotypes associated with the complex I assembly defect. Our work shows that CG7598/dCIA30 is a functional homolog of Ndufaf1 and adds to the accumulating evidence that transgenic NDI1 expression is a viable therapy for disorders arising from complex I deficiency.     

Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.     

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the CuA center of cytochrome c oxidase

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the Cu_A center contained within subunit II of the oxidase complex. The Cu_A center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a₃ and Cu_B. Cu_A consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of Cu_A present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in Cu_A assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-Cu_A site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Sucrose and glycerol effects on photosystem II

Photosystem II catalyzes the oxidation of water and the reduction of plastoquinone. The active site cycles among five oxidation states, which are called the S(n) states. PSII purification procedures include the use of the cosolvents, sucrose and/or glycerol, to stabilize water splitting activity and for cryoprotection. In this study, the effects of sucrose and glycerol on PSII were investigated. Sucrose addition was observed to stimulate the steady-state rate of oxygen evolution in the range from 0 to 1.35 M. Glycerol addition was observed to stimulate oxygen evolution in the range from 0 to 30%. Both cosolvents were observed to be inhibitory at higher concentrations. Sucrose addition was shown to have no effect on the rate of Q(A)(-) oxidation or on the K(M) for exogenous acceptor. PSII was then treated to remove extrinsic proteins. In these samples, sucrose addition stimulated activity, but glycerol addition was inhibitory at concentrations higher than approximately 0.5 M. This inhibitory effect of glycerol at relatively low concentrations is attributed to glycerol binding to the active site, when extrinsic subunits are not present. Reaction induced FTIR spectra, associated with the S(1) to S(2) transition of the water-oxidizing complex, exhibited significant differences throughout the 1,800-1,200 cm(-1) region, when glycerol- and sucrose-containing samples were compared. These measurements suggest a cosolvent-induced shift in the pK(A) of an aspartic or glutamic acid side chain, as well as structural changes at the active site. These structural alterations are attributed to a change in preferential hydration of the oxygen-evolving complex.     

Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg²⁺ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.     

Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction ν, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction ν that cleaves VAMP2, and report its amino acid sequence.     

Copper binding traps the folded state of the SCO protein from Bacillus subtilis

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu_A center. BsSCO has been proposed to play various roles in Cu_A assembly including, the direct delivery of copper ions to the Cu_A site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu_A. BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.     

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO : Relationship to Large Subunit-Binding Protein Complex Dissociation

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.