RESUMO
Mitochondria play a pivotal part in ATP energy production through oxidative phosphorylation, which occurs within the inner membrane through a series of respiratory complexes1-4. Despite extensive in vitro structural studies, determining the atomic details of their molecular mechanisms in physiological states remains a major challenge, primarily because of loss of the native environment during purification. Here we directly image porcine mitochondria using an in situ cryo-electron microscopy approach. This enables us to determine the structures of various high-order assemblies of respiratory supercomplexes in their native states. We identify four main supercomplex organizations: I1III2IV1, I1III2IV2, I2III2IV2 and I2III4IV2, which potentially expand into higher-order arrays on the inner membranes. These diverse supercomplexes are largely formed by 'protein-lipids-protein' interactions, which in turn have a substantial impact on the local geometry of the surrounding membranes. Our in situ structures also capture numerous reactive intermediates within these respiratory supercomplexes, shedding light on the dynamic processes of the ubiquinone/ubiquinol exchange mechanism in complex I and the Q-cycle in complex III. Structural comparison of supercomplexes from mitochondria treated under different conditions indicates a possible correlation between conformational states of complexes I and III, probably in response to environmental changes. By preserving the native membrane environment, our approach enables structural studies of mitochondrial respiratory supercomplexes in reaction at high resolution across multiple scales, from atomic-level details to the broader subcellular context.
Assuntos
Respiração Celular , Complexo III da Cadeia de Transporte de Elétrons , Complexo I de Transporte de Elétrons , Mitocôndrias , Animais , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/ultraestrutura , Modelos Moleculares , Fosforilação Oxidativa , Suínos , Ubiquinona/análogos & derivados , Ubiquinona/química , Ubiquinona/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismoRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Complexo III da Cadeia de Transporte de Elétrons , Complexo II de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Complexo I de Transporte de Elétrons , Mitocôndrias , Membranas Mitocondriais , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/ultraestrutura , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Complexo II de Transporte de Elétrons/química , Complexo II de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/ultraestrutura , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Simulação de Dinâmica Molecular , Sítios de Ligação , Evolução MolecularRESUMO
Proteins destined to various intra- and extra-cellular locations must traverse membranes most frequently in an unfolded form. When the proteins being translocated need to remain in a folded state, specialized cellular transport machinery is used. One such machine is the membrane-bound AAA protein Bcs1 (Bcs1), which assists the iron-sulfur protein, an essential subunit of the respiratory Complex III, across the mitochondrial inner membrane. Recent structure determinations of mouse and yeast Bcs1 in three different nucleotide states reveal its homo-heptameric association and at least two dramatically different conformations. The apo and ADP-bound structures are similar, both containing a large substrate-binding cavity accessible to the mitochondrial matrix space, which contracts by concerted motion of the ATPase domains upon ATP binding, suggesting that bound substrate could then be pushed across the membrane. ATP hydrolysis drives substrate release and resets Bcs1 conformation back to the apo/ADP form. These structures shed new light on the mechanism of folded protein translocation across a membrane, provide better understanding on the assembly process of the respiratory Complex III, and correlate clinical presentations of disease-associated mutations with their locations in the 3D structure.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/genética , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/genética , Animais , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/ultraestrutura , Camundongos , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Domínios Proteicos/genética , Dobramento de Proteína , Transporte Proteico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestruturaAssuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/ultraestrutura , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestrutura , Estrutura Secundária de ProteínaRESUMO
Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria1-3. Like complex III (also known as the bc1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity4-7. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily 8 , including the proton pump polysulfide reductase9,10. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer11-14, both as an independent enzyme and as a functional 1:1 supercomplex with an aa3-type cytochrome c oxidase (cyt aa3). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.
Assuntos
Microscopia Crioeletrônica , Grupo dos Citocromos c/química , Grupo dos Citocromos c/ultraestrutura , Citocromos a3/química , Citocromos a3/ultraestrutura , Citocromos a/química , Citocromos a/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Flavobacterium/enzimologia , Cisteína/química , Cisteína/metabolismo , Grupo dos Citocromos c/metabolismo , Citocromos a/metabolismo , Citocromos a3/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Heme/análogos & derivados , Heme/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos/química , Modelos Moleculares , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
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.
Assuntos
Respiração Celular , Microscopia Crioeletrônica , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/ultraestrutura , Complexo I de Transporte de Elétrons/ultraestrutura , Animais , Sítios de Ligação , Domínio Catalítico , Transporte de Elétrons , Complexo I de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Coração , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , OvinosRESUMO
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.
Assuntos
Cristalização/métodos , Detergentes/química , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Polímeros/química , Propilaminas/química , Tensoativos/química , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Dobramento de Proteína , Solubilidade , Soluções , Água/químicaRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Tomografia/métodos , Animais , Bovinos , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/ultraestrutura , Modelos Moleculares , Ligação ProteicaRESUMO
Propamidine, an aromatic diamidine compound, is widely used as an antimicrobial agent. To uncover its mechanism on pathogenetic fungi, Botrytis cinerea as an object was used to investigate effects of propamidine in this paper. The transmission electron microscope results showed that the mitochondrial membranes were collapsed after propamidine treatment, followed that mitochondria were disrupted. Inhibition of whole-cell and mitochondrial respiration by propamidine suggested that Propamidine is most likely an inhibitor of electron transport within Botrytis cinerea mitochondria. Furthermore, the mitochondrial complex III activity were inhibited by propamidine.
Assuntos
Benzamidinas/farmacologia , Botrytis/efeitos dos fármacos , Botrytis/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Botrytis/ultraestrutura , Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Microscopia Eletrônica de Transmissão , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestruturaRESUMO
The bacterium Blastochloris viridis carries one of the simplest photosynthetic systems, which includes a single light-harvesting complex that surrounds the reaction center, membrane soluble quinones, and a soluble periplasmic protein cytochrome c(2) that shuttle between the reaction center and the bc(1) complex and act as electron carriers, as well as the ATP synthase. The close arrangement of the photosynthetic membranes in Bl. viridis, along with the extremely tight arrangement of the photosystems within these membranes, raises a fundamental question about the diffusion of the electron carriers. To address this issue, we analyzed the structure and response of the Bl. viridis photosynthetic system to various light conditions, by using a combination of electron microscopy, whole-cell cryotomography, and spectroscopic methods. We demonstrate that in response to high light intensities, the ratio of both cytochrome c(2) and bc(1) complexes to the reaction centers is increased. The shorter membrane stacks, along with the notion that the bc(1) complex is located at the highly curved edges of these stacks, result in a smaller average distance between the reaction centers and the bc(1) complexes, leading to shorter pathways of cytochrome c(2) between the two complexes. Under anaerobic conditions, the slow diffusion rate is further mitigated by keeping most of the quinone pool reduced, resulting in a concentration gradient of quinols that allows for a constant supply of theses electron carriers to the bc(1) complex.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Rodopseudomonas/química , Adaptação Fisiológica , ATPases Bacterianas Próton-Translocadoras/química , ATPases Bacterianas Próton-Translocadoras/ultraestrutura , Difusão , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Fluorometria , Cinética , Luz , Fotoquímica , Complexo de Proteínas do Centro de Reação Fotossintética/ultraestrutura , Rodopseudomonas/enzimologia , Rodopseudomonas/crescimento & desenvolvimento , Rodopseudomonas/ultraestrutura , Tilacoides/química , Tilacoides/enzimologia , Tilacoides/ultraestruturaRESUMO
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.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Modelos Químicos , Modelos Moleculares , Rhodobacter capsulatus/enzimologia , Zinco/química , Animais , Sítios de Ligação , Aves , Bovinos , Simulação por Computador , Ligação Proteica , Especificidade da EspécieRESUMO
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.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Animais , Bovinos , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/ultraestrutura , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Mitocôndrias Cardíacas/metabolismo , Modelos Moleculares , Complexos MultiproteicosRESUMO
Mitochondria are central to the efficient provision of energy for eukaryotic cells. The oxidative-phosphorylation system of mitochondria consists of a series of five major membrane complexes: NADH-ubiquinone oxidoreductase (commonly known as complex I), succinate-ubiquinone oxidoreductase (complex II), ubiquinol-cytochrome c oxidoreductase (cytochrome bc1 complex or complex III), cytochrome c-O2 oxidoreductase (complex IV), and F1F0-ATP synthase (complex V). Several lines of evidence have recently suggested that complexes I and III-V might interact to form supercomplexes. However, because of their fragility, the structures of these supercomplexes are still unknown. A stable supercomplex consisting of complex I and dimeric complex III was purified from plant mitochondria. Structural characterization by single-particle EM indicates a specific type of interaction between monomeric complex I and dimeric complex III in a 1:1 ratio. We present a model for how complexes I and III are spatially organized within the I+III2 supercomplex.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Arabidopsis , Transporte de Elétrons , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/isolamento & purificação , Complexo I de Transporte de Elétrons/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/isolamento & purificação , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , Modelos Moleculares , Conformação ProteicaRESUMO
Cytochrome bc1 complex from bovine heart has been reconstituted into tubular crystals. The three-dimensional structure of the complex in lipid bilayer has been obtained at an effective resolution of 16 angstrom by electron cryomicroscopy and helical image reconstruction. The complex is in a dimeric form, in which the monomers are associated closely in extramembrane domains on both sides of the membrane. The large inner domain is distinctively hollow and the small outer domain consists of a flat mass and two bulbous extrusions. These domains are connected by two narrow transmembrane columns. Locations of the subunits and the redox centres in the model are proposed.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/química , Animais , Sítios de Ligação , Bovinos , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Simulação por Computador , Cristalização , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Congelamento , Bicamadas Lipídicas , Microscopia Eletrônica , Modelos Moleculares , Neurospora/enzimologia , Oxirredução , Ubiquinona/metabolismoRESUMO
Ubiquinol-cytochrome c reductase is a crucial integral membrane protein in the mitochondrial respiratory cycle. Eleven subunits containing three cytochrome heme groups and a 2Fe-2S Rieske center make up this 240 kDa enzyme complex. Previously, many different crystal forms of the bc1 complex have displayed diffraction to as far as 4.5 A. However, rapid degradation of the protein in the X-ray beam at room temperature has obstructed the collection of a full data set from a single crystal. As slight heterogeneities between crystals severely hampered merging of data from different crystals, we sought a method to stabilize the protein crystal in the X-ray beam in order to collect a full data set from one crystal sample. To this end, water soluble protein crystals are frequently flash-cooled to cryogenic temperatures; however, there is no report of cryocrystallography for membrane proteins. In this communication, we report on a successful experiment in which flash-cooled bc1 membrane protein crystals have given rise to sustained diffraction over a 60 hour data collection period at a synchrotron source. Furthermore, we present an improved purification and crystallization protocol yielding crystals readily diffracting out to 3.3 A. These results should greatly aid in the future realization of the molecular structure of the bc1 complex as well as other membrane proteins.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Proteínas de Membrana/ultraestrutura , Animais , Bovinos , Cristalografia por Raios X , Mitocôndrias CardíacasRESUMO
The topographical localization of the N-terminus of cytochrome b in the inner mitochondrial membrane was determined by mild proteolysis of the yeast mitochondrial cytochrome bc1 complex and identification of the proteolytic fragments derived from subunits of the complex with an established orientation in the inner membrane. The cytochrome bc1 complex was incorporated into proteoliposomes which were separated by cytochrome c affinity chromatography into two populations in either the mitochondrial or the submitochondrial orientation. Core protein I which protrudes from the matrix side of the inner membrane was digested by proteinase K only in proteoliposomes with the submitochondrial orientation and not in those with the mitochondrial orientation. By contrast, cytochrome c1 with protrudes from the cytoplasmic side of the inner membrane was digested by proteinase K only in proteoliposomes with the mitochondrial orientation and not in those with the submitochondrial orientation. Cytochrome b was digested by SV8 protease only in proteoliposomes with the mitochondrial orientation to yield two aggregating fragments of 25.6 and 24.5 kDa. These peptides were isolated by preparative gel chromatography and sequenced to establish that the cleavage of cytochrome b by SV8 protease occurred at glutamate residues 59 and 66. These residues are localized in the extramembranous loop between the two hydrophobic membrane-spanning helices A and B and thus face the cytoplasmic side of the inner mitochondrial membrane. These results indicate that the N-terminus of yeast cytochrome b protrudes from the matrix side of the inner membrane consistent with the eight-helix model for the orientation of cytochrome b in the membrane.
Assuntos
Grupo dos Citocromos b/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Membranas Intracelulares/ultraestrutura , Mitocôndrias/ultraestrutura , Leveduras/ultraestrutura , Sequência de Aminoácidos , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Endopeptidase K , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteolipídeos , Análise de Sequência , Serina Endopeptidases/metabolismoRESUMO
Beef heart mitochondrial ubiquinol:cytochrome c oxidoreductase has been crystallized in the shape of hexagonal bipyramids. At present the crystals diffract X-rays to 4.7 A. From preliminary analysis the diffraction pattern appears to be consistent with space group P6(1)22 or P6(5)22 and with unit cell parameters a = b = 212 A and c = 352 A.