Structural basis of mitochondrial membrane bending by the I-II-III2-IV2 supercomplex.

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.

Allosteric Cooperativity in Proton Energy Conversion in A1-Type Cytochrome c Oxidase.

Cytochrome c oxidase (CcO), the CuA, heme a, heme a3, CuB enzyme of respiratory chain, converts the free energy released by aerobic cytochrome c oxidation into a membrane electrochemical proton gradient (ΔµH+). ΔµH+ derives from the membrane anisotropic arrangement of dioxygen reduction to two water molecules and transmembrane proton pumping from a negative (N) space to a positive (P) space separated by the membrane. Spectroscopic, potentiometric, and X-ray crystallographic analyses characterize allosteric cooperativity of dioxygen binding and reduction with protonmotive conformational states of CcO. These studies show that allosteric cooperativity stabilizes the favorable conformational state for conversion of redox energy into a transmembrane ΔµH+.

Alterations of complex IV in the tissues of a septic mouse model.

OBJECTIVES: To characterize the mitochondrial respiratory chain complex IV(complex IV) activity and protein expression during polymicrobial sepsis. MATERIAL AND METHODS: Polymicrobial peritonitis, a clinically relevant mouse model of sepsis, was generated by cecum ligation and puncture (CLP) in Sprague- Dawley rats. The rats were randomly divided into 3 groups as follows: the sepsis without resuscitation (S), sepsis and fluid resuscitated (R) group, and a control (C) group. Twelve hours after the sepsis model was established, tissue specimens were obtained from the myocardium, liver and skeletal muscle. Mitochondrial respiratory chain complex IV activity of all tissue specimens was detected by spectrophotometry. Western blot was used to measure the liver mitochondrial respiratory chain complex IV protein content. The ultrastructure changes of mitochondria were detected by transmission electron microscopy. RESULTS: In myocardial cells, complex IV activity decreased significantly in the S and R groups as compared to the C group. There were no differences in complex IV activity between groups in skeletal muscle cells while in liver cells, complex IV activity and content was significantly decreased for the S group but no differences were observed between the C and R groups. Increased matrix volume and reduced density with generalized disruption of the normal cristae pattern was most extensive in the liver, followed by cardiac muscle cells with that in skeletal muscle cells been relatively mild in the S group. Mitochondrial fusion/fission and mitochondrial autophagy was also observed in the S group by transmission electron microscopy. Mitochondrial ultrastructure was preserved in the R-group and was similar to that seen in the C-group. CONCLUSIONS: Changes in complex IV activity and mitochondrial ultrastructure, a manifestation of the mitochondrial dysfunction varied depending on cell type. These changes are partly reversed by fluid therapy. Therapies aimed at mitochondrial resuscitation should be explored.

On the role of subunit M in cytochrome cbb3 oxidase.

Cytochrome cbb3 (or C-type) oxidases are a highly divergent group and the least studied members of the heme-copper oxidases (HCOs) superfamily. HCOs couple the reduction of oxygen at the end of the respiratory chain to the active proton translocation across the membrane, contributing to establishment of an electrochemical gradient essential for ATP synthesis. Cbb3 oxidases exhibit unique structural and functional features and have an essential role in the metabolism of many clinically relevant human pathogens. Such characteristics make them a promising therapeutic target. Three subunits, N, O and P, comprise the core cbb3 complex, with N, the catalytic subunit, being highly conserved among all members of the HCO superfamily, including the A-type (aa3, mitochondrial-like) oxidases. An additional fourth subunit containing a single transmembrane (TM) helix was present in the first crystal structure of cbb3. This TM segment was recently proposed to be part of a novel protein CcoM, which was shown to have a putative role in the complex stability and assembly. In this work, we performed large-scale all-atom molecular dynamics simulations of the CcoNOPM complex to further characterize the interactions between subunit M and the core subunits and to determine whether the presence of the fourth subunit influences the water/proton channels previously described for the core complex. The previously proposed putative CcoNOPH complex is also assessed, and the potential functional redundancy of CcoM and CcoQ is discussed.

The architecture of respiratory supercomplexes.

Mitochondrial electron transport chain complexes are organized into supercomplexes responsible for carrying out cellular respiration. Here we present three architectures of mammalian (ovine) supercomplexes determined by cryo-electron microscopy. We identify two distinct arrangements of supercomplex CICIII2CIV (the respirasome)-a major 'tight' form and a minor 'loose' form (resolved at the resolution of 5.8 Å and 6.7 Å, respectively), which may represent different stages in supercomplex assembly or disassembly. We have also determined an architecture of supercomplex CICIII2 at 7.8 Å resolution. All observed density can be attributed to the known 80 subunits of the individual complexes, including 132 transmembrane helices. The individual complexes form tight interactions that vary between the architectures, with complex IV subunit COX7a switching contact from complex III to complex I. The arrangement of active sites within the supercomplex may help control reactive oxygen species production. To our knowledge, these are the first complete architectures of the dominant, physiologically relevant state of the electron transport chain.

Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases.

The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper-containing oxidases, even though the structures are completely different.

From static structure to living protein: computational analysis of cytochrome c oxidase main-chain flexibility.

Crystallographic structure and deuterium accessibility comparisons of CcO in different redox states have suggested conformational changes of mechanistic significance. To predict the intrinsic flexibility and low energy motions in CcO, this work has analyzed available high-resolution crystallographic structures with ProFlex and elNémo computational methods. The results identify flexible regions and potential conformational changes in CcO that correlate well with published structural and biochemical data and provide mechanistic insights. CcO is predicted to undergo rotational motions on the interior and exterior of the membrane, driven by transmembrane helical tilting and bending, coupled with rocking of the ß-sheet domain. Consequently, the proton K-pathway becomes sufficiently flexible for internal water molecules to alternately occupy upper and lower parts of the pathway, associated with conserved Thr-359 and Lys-362 residues. The D-pathway helices are found to be relatively rigid, with a highly flexible entrance region involving the subunit I C-terminus, potentially regulating the uptake of protons. Constriction and dilation of hydrophobic channels in RsCcO suggest regulation of the oxygen supply to the binuclear center. This analysis points to coupled conformational changes in CcO and their potential to influence both proton and oxygen access.

Interaction of complexes I, III, and IV within the bovine respirasome by single particle cryoelectron tomography.

The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III(2), and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III(2) and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening.

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ∼60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 µM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.

Electron and proton transfer in the ba(3) oxidase from Thermus thermophilus.

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa(3)-type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O(2) to water is catalysed at a haem a(3)-Cu(B) catalytic site. The three-dimensional structure of the ba(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa(3) oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba( 3 )-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k congruent with 6.8 x 10(4) s(-1)) and formation of the peroxy (P(R)) intermediate. In the next step a proton was taken up from solution with a rate constant of approximately 1.7 x 10(4) s(-1), associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of approximately 1,100 s(-1), accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was approximately 1.5 protons per enzyme molecule. The results support the earlier proposal that the P(R) and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba(3) oxidases, the proton-uptake reactions occur over the same time scales as in the aa(3)-type oxidases.

Thermostability of proteins: role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus.

The dinuclear copper center (TtCuA) forming the electron entry site in the subunit II of the cytochrome c oxidase in Thermus thermophilus shows high stability toward thermal as well as denaturant-induced unfolding of the protein at ambient pH. We have studied the effect of pH on the stability of the holo-protein as well as of the apo-protein by UV-visible absorption, far-UV, and visible circular dichroism spectroscopy. The results show that the holo-protein both in the native mixed-valence state as well as in the reduced state of the metal ions and the apo-protein of TtCuA were extremely stable toward unfolding by guanidine hydrochloride at ambient pH. The thermal unfolding studies at different values of pH suggested that decreasing pH had almost no effect on the thermal stability of the protein in the absence of the denaturant. However, the stability of the proteins in presence of the denaturant was considerably decreased on lowering the pH. Moreover, the stability of the holo-protein in the reduced state of the metal ion was found to be lower than that in the mixed-valence state at the same pH. The denaturant-induced unfolding of the protein at different values of pH was analyzed using a two-state unfolding model. The values of the free energy of unfolding were found to increase with pH. The holo-protein showed that the variation of the unfolding free energy was associated with a pKa of approximately 5.5. This is consistent with the model that the protonation of a histidine residue may be responsible for the decrease in the stability of the holo-protein at low pH. The results were interpreted in the light of the reported crystal structure of the protein.

Architecture of active mammalian respiratory chain supercomplexes.

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.

Properties of the detergent solubilised cytochrome c oxidase (cytochrome cbb(3)) purified from Pseudomonas stutzeri.

Cytochrome cbb(3) is a cytochrome c-oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases. We have developed a purification method yielding large amounts of pure cbb(3) complex from the soil bacterium Pseudomonas stutzeri. This cytochrome cbb(3) complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b-type and three c-type haems. The protein complex behaves as a monomer with an overall molecular weight of 114.0+/-8.9 kDa and a s(0)(20,w) value of 8.9+/-0.3 S as determined by analytical ultracentrifugation. Crystals diffracting to 5.0 A resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1 x 0.1 x 0.3 mm. This is the first crystallisation report of a (cbb(3))-type oxidase.

Chorion laeve trophoblasts of preeclamptic fetal membranes: histochemically detectable enzyme activities do not change at a subcellular level.

We examined the subcellular localization of ADP-degrading activity and cytochrome c oxidase (CCO) activity in chorion laeve trophoblasts from term and near term human fetal membranes, and compared them with those from severe preeclamptic fetal membranes. The methods used for the detection of enzyme activities were the lead nitrate method for ADP-degrading activity and the diaminobenzidine method for CCO. Precipitates indicative of ADP-degrading activity were visible on surface microvillous plasma membranes of chorion laeve trophoblasts both from normal and preeclamptic fetal membranes. The intensity and distribution patterns were the same in the normal and preeclamptic subjects. CCO labeling was visible in almost all laeve trophoblastic mitochondria both in normal and preeclamptic cases. Previously, we demonstrated that in preeclamptic villous trophoblasts there were decreases in ADP-degrading activity and the presence of CCO-negative mitochondria, which were proposed to lead to dysfunction of each villous trophoblast, and finally to placental insufficiency in preeclampsia. Reductions or changes in enzyme intensities/distribution patterns, which are characteristic features of preeclamptic villous trophoblasts, were absent in chorion laeve trophoblasts in preeclampsia. These results suggest that in preeclampsia there are no, or at least less severe, abnormalities in the enzyme activities of chorion laeve trophoblasts, compared with villous trophoblasts, as far as enzyme-histochemically detectable enzymes are concerned.

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.

Brain cytochrome oxidase subunit complementary DNAs: isolation, subcloning, sequencing, light and electron microscopic in situ hybridization of transcripts, and regulation by neuronal activity.

The goal of the present study was to isolate, for the first time, cytochrome oxidase subunit genes from murine brain complementary DNA library and to characterize the expression of these genes from mitochondrial and nuclear sources at both light and electron microscopic levels. Brain subunit III (mitochondrial) shared 100% identity with that of murine L cells. Subunit VIa (nuclear) was known to have tissue-specific isoforms in other species: the ubiquitous liver isoform and the heart/muscle isoform. Our brain subunit VIa shared 93% homology with that of the rat liver and 100% identity with the recently reported murine liver isoform, which is only 62% identical to that of the rat heart isoform. In situ hybridization with riboprobes revealed messenger RNA labelling that was similar, though not identical, to that of cytochrome oxidase histochemistry. Monocular enucleation in adult mice induced a significant down-regulation of both subunit messages in the contralateral lateral geniculate nucleus. However, the decrease in subunit III messenger RNAs surpassed that of subunit VIa at all time periods examined, suggesting that mitochondrial gene expression is more tightly regulated by neuronal activity than that of nuclear ones. At the electron microscopic level, subunit III messenger RNA was localized to the mitochondrial compartment in both cell bodies and processes, while that of nuclear-encoded subunit VIa was present exclusively in the extramitochondrial compartment of somata and not of dendrites or axons. Surprisingly, the message was primarily associated with the rough endoplasmic reticulum, suggesting a novel pathway for its synthesis and trafficking. Our results indicate that the unique properties of neurons impose special requirements for subunits of a single mitochondrial enzyme with dual genomic origins. At sites of high energy demands (such as postsynaptic dendrites and some axon terminals), mitochondrial-encoded cytochrome oxidase subunits can be locally transcribed and translated, and they provide the framework for the subsequent importation and incorporation of nuclear-encoded subunits, which are strictly synthesized in the cell bodies. Dynamic local energy needs are met when subunits from the two genomic sources are assembled to form functional holoenzymes.

Multiple mitochondrial DNA deletions in sporadic inclusion body myositis: a study of 56 patients.

Inclusion body myositis, a chronic inflammatory disorder, is the most common cause of myopathy in adults over the age of 50. Diagnosis is based on clinical features and distinctive morphological findings by both light and electron microscopy. The causes of inclusion body myositis are still unknown. Ultrastructural mitochondrial changes and ragged-red fibers are common in patients with sporadic inclusion body myositis, and multiple [correction of mutiple] mitochondrial DNA (mtDNA) deletions have been reported in 3 such patients, suggesting that mtDNA mutations may have a pathogenetic role. We studied 56 patients with sporadic inclusion body myositis, using a combination of clinical, morphological, biochemical, and molecular genetic analyses to determine the frequency and the distribution of mtDNA deletions. Using the polymerase chain reaction, we found multiple mtDNA deletions in 73% of patients, compared to 40% of normal age-matched control subjects and 47% of disease control subjects. The presence of deletions correlated with morphological evidence of ragged-red, cytochrome c oxidase-negative fibers, and with defects of complexes I and IV of the electron transport chain. Although aging may account for a proportion of mtDNA deletions in patients with sporadic inclusion body myositis and control subjects, mtDNA alterations may be accelerated in sporadic inclusion body myositis.

Reversed metal replicas of freeze-dried proteins to be visualized with the scanning tunneling microscope.

Scanning tunneling microscopy of metal-coated specimens has become a reliable technique that permits direct three-dimensional visualization of structural details at a level at which individual subunits in protein complexes or even single domains of proteins can be resolved. We describe in this paper a variation of the freeze-drying metal coating procedure that allows us to image with the STM the inner side of the metal replica, previously in contact with the protein molecules. We have tested this new approach with two different well characterized protein systems: freeze-dried two-dimensional crystals of bacteriophage phi 29 connector and the vesicle form of two-dimensional crystals of cytochrome oxidase from beef heart mitochondria. The images obtained have very good contrast and provide direct topographic information of the crystal surface, complementing structural information obtained previously with transmission electron microscopy. The resolution limit is imposed by the size (2-3 nm diameter) and corrugation of the metal grains used to prepare the replica and by the randomness of the metal shadowing.

Site-directed mutagenesis of residues within helix VI in subunit I of the cytochrome bo3 ubiquinol oxidase from Escherichia coli suggests that tyrosine 288 may be a CuB ligand.

The heme-copper oxidase superfamily contains all of the mammalian mitochondrial cytochrome c oxidases, as well as most prokaryotic respiratory oxidases. All members of the superfamily have a subunit homologous to subunit I of the mammalian cytochrome c oxidases. This subunit provides the amino acid ligands to a low-spin heme component as well as to a heme-copper binuclear center, which is the site where dioxygen is reduced to water. The amino acid sequence of transmembrane helix VI of subunit I is the most highly conserved within the superfamily. Previous efforts have demonstrated that one of the residues in this region, H284, is critical for oxidase activity and for the assembly of CuB. This paper presents the analysis of additional site-directed mutants in which other highly conserved residues in helix VI (P285, E286, Y288, and P293) have been substituted. Most of the mutants are enzymatically inactive. Structural perturbations reported by Fourier transform infrared absorption difference spectroscopy of CO adducts of the mutant oxidases confirm the previous suggestion that this region is adjactent to CuB. Furthermore, the analysis of five different substitutions for Y288 indicates that all lack CuB. On the basis of these data, it is proposed that Y288 may be a CuB ligand along with H333, H334, and H284, and a plausible molecular model of the CuB site is presented.

Electron microscopy of cytochrome c oxidase crystals: labeling of subunit III with a monomaleimide undecagold cluster compound.

Two-dimensional crystals of beef heart mitochondrial cytochrome c oxidase dimers were labeled at Cys-115 of subunit III with a monomaleimide derivative of an undecagold cluster compound. The binding site of the gold cluster compound and hence the site of subunit III were identified by image processing of cryoelectron micrographs of the crystals preserved in a mixture of glucose and uranyl acetate. The shape of the cytochrome oxidase dimer can be approximated as a parallelogram which is 44 by 82 A with an included angle of 80 degrees oriented with its long dimension along the a axis of the crystal. Labeling of subunit III was confirmed by a shift in the mobility of approximately 50% of subunit III molecules upon electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. Averaged images of undecagold cluster labeled crystals and of unlabeled crystals were calculated; each image represents an average of approximately 17,000 molecules of either labeled or unlabeled cytochrome oxidase. On the basis of a statistical analysis of the differences between the two images, the gold cluster binds along a line 30 degrees from the a axis and 29 A from the center of the dimer. This result is interpreted in the context of other structural studies including the site of cytochrome c binding which Frey and Murray found to be near the a axis and 18 A from the center of the dimer [Frey, T. G., & Murray, J. M. (1994) J. Mol. Biol. 237, 275-297].(ABSTRACT TRUNCATED AT 250 WORDS)