The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria

The mitochondrial electron transport chain complexes are large multisubunit complexes embedded in the inner membrane. We report here that in the yeast Saccharomyces cerevisiae, the cytochrome bc(1) and cytochrome c oxidase complexes co-exist as a larger complex of approximately 1000 kDa in the mitochondrial membrane. Following solubilization with a mild detergent, the cytochrome bc(1)-cytochrome c oxidase complex remains stable. It was analyzed using the techniques of gel filtration and blue native-polyacrylamide gel electrophoresis. Direct physical association of subunits of the cytochrome bc(1) complex with those of the cytochrome c oxidase complex was verified by co-immunoprecipitation analysis. Our data indicate that the cytochrome bc(1) complex is exclusively in association with the cytochrome c oxidase complex in yeast mitochondria. We term this complex the cytochrome bc(1)-cytochrome c oxidase supracomplex.     

Biogenesis of the yeast cytochrome bc1 complex

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

The stoichiometry of charge translocation by cytochrome oxidase and the cytochrome bc1 complex of mitochondria at high membrane potential

The q+/2e stoichiometries (number of charges translocated per electron pair transferred) of cytochrome oxidase and the cytochrome bc1 complex in rat liver mitochondria were determined at a range of membrane potentials up to 180 mV. The method used was similar to the one used in the preceding paper by us in this journal to determine the q+/O stoichiometry of the mitochondrial electron transport chain from succinate to oxygen. The measured q+/2e stoichiometry of cytochrome oxidase was 3.5 positive charges per O atom reduced at low membrane potential (120 mV) and it decreased to about 1.5 at high membrane potential (180 mV). The measured q+/2e stoichiometry of the cytochrome bc1 complex was between 1 and 1.25 positive charges ejected per electron pair and did not change significantly as delta psi was varied from 85 mV to 157 mV. The sum of the q+/2e stoichiometries of cytochrome oxidase and the cytochrome bc1 complex determined separately was similar to their value determined together for electron transport from succinate to oxygen over the range of membrane potentials studied. The most probable interpretation of these results is that the stoichiometry of the cytochrome bc1 complex is invariant over a range of membrane potentials and that the q+/2e stoichiometry of cytochrome oxidase decreases from 4 at low membrane potential to 2 at high membrane potential.     

A yeast mitochondrial inner membrane 30K hydrophobic protein: comparison with subunit 32K of the cytochrome BC 1 complex

A yeast mitochondrial inner membrane hydrophobic protein 30K has been isolated and compared to subunit 32K of the yeast cytochrome bc 1 complex. Both proteins are translated on mitochondrial ribosomes, have nearly the same molecular weight and similar aminoacid compositions. Comparison was carried out by immunological techniques with specific antibodies, and by studying 3 yeast strains having mutations in the COB region of the mitochondrial DNA. Results show that the two proteins are not identical.     

Protonmotive pathways and mechanisms in the cytochrome bc1 complex

The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.     

Identification and characterization of cytochrome bc(1) subcomplexes in mitochondria from yeast with single and double deletions of genes encoding cytochrome bc(1) subunits

We have examined the status of the cytochrome bc(1) complex in mitochondrial membranes from yeast mutants in which genes for one or more of the cytochrome bc(1) complex subunits were deleted. When membranes from wild-type yeast were resolved by native gel electrophoresis and analyzed by immunodecoration, the cytochrome bc(1) complex was detected as a mixed population of enzymes, consisting of cytochrome bc(1) dimers, and ternary complexes of cytochrome bc(1) dimers associated with one and two copies of the cytochrome c oxidase complex. When membranes from the deletion mutants were resolved and analyzed, the cytochrome bc(1) dimer was not associated with the cytochrome c oxidase complex in many of the mutant membranes, and membranes from some of the mutants contained a common set of cytochrome bc(1) subcomplexes. When these subcomplexes were fractionated by SDS/PAGE and analyzed with subunit-specific antibodies, it was possible to recognize a subcomplex consisting of cytochrome b, subunit 7 and subunit 8 that is apparently associated with cytochrome c oxidase early in the assembly process, prior to acquisition of the remaining cytochrome bc(1) subunits. It was also possible to identify a subcomplex consisting of subunit 9 and the Rieske protein, and two subcomplexes containing cytochrome c(1) associated with core protein 1 and core protein 2, respectively. The analysis of all the cytochrome bc(1) subcomplexes with monospecific antibodies directed against Bcs1p revealed that this chaperone protein is involved in a late stage of cytochrome bc(1) complex assembly.     

The iron-sulfur protein of cytochrome bc1 complex. Its occurrence in the mitochondrial inner membrane in excess of the amount constituting the complex

Radioimmunoassay and quantitative immunoblot analysis have been developed for quantitation of the iron-sulfur protein of cytochrome bc1 complex in order to compare its content in isolated cytochrome bc1 complex with that in electron transport particles. The result by radioimmunoassay indicated that the content of the iron-sulfur protein/mol of cytochrome b is higher by approximately 30%, on the average, in electron transport particles than in cytochrome bc1 complex. This observation was supported by the data of immunoblot analysis. Since approximately 1/3 of cytochrome b in electron transport particles is not attributed to cytochrome bc1 complex, but to succinate-ubiquinone oxidoreductase complex (Davis, K.A., Hatefi, Y., Poff, K. L., and Butler, W. L. (1973) Biochim. Biophys. Acta 325, 341-356), the ratio of the iron-sulfur protein detectable by radioimmunoassay in electron transport particles to that in cytochrome bc1 complex is calculated to be approximately 2 on the basis of the content of 2 mol of b-type heme/mol of the complex. Therefore, it appears that the mitochondrial inner membrane contains approximately two times as much of the immunoreactive iron-sulfur protein as what is expected from the stoichiometry of one iron-sulfur center and two b-type hemes for cytochrome bc1 complex. This finding affords an interesting aspect in the study of biogenesis of cytochrome bc1 complex.     

Cytochrome bc1 complexes of microorganisms.

The cytochrome bc1 complex is the most widely occurring electron transfer complex capable of energy transduction. Cytochrome bc1 complexes are found in the plasma membranes of phylogenetically diverse photosynthetic and respiring bacteria, and in the inner mitochondrial membrane of all eucaryotic cells. In all of these species the bc1 complex transfers electrons from a low-potential quinol to a higher-potential c-type cytochrome and links this electron transfer to proton translocation. Most bacteria also possess alternative pathways of quinol oxidation capable of circumventing the bc1 complex, but these pathways generally lack the energy-transducing, protontranslocating activity of the bc1 complex. All cytochrome bc1 complexes contain three electron transfer proteins which contain four redox prosthetic groups. These are cytochrome b, which contains two b heme groups that differ in their optical and thermodynamic properties; cytochrome c1, which contains a covalently bound c-type heme; and a 2Fe-2S iron-sulfur protein. The mechanism which links proton translocation to electron transfer through these proteins is the proton motive Q cycle, and this mechanism appears to be universal to all bc1 complexes. Experimentation is currently focused on understanding selected structure-function relationships prerequisite for these redox proteins to participate in the Q-cycle mechanism. The cytochrome bc1 complexes of mitochondria differ from those of bacteria, in that the former contain six to eight supernumerary polypeptides, in addition to the three redox proteins common to bacteria and mitochondria. These extra polypeptides are encoded in the nucleus and do not contain redox prosthetic groups. The functions of the supernumerary polypeptides of the mitochondrial bc1 complexes are generally not known and are being actively explored by genetically manipulating these proteins in Saccharomyces cerevisiae.     

Pet191 is a cytochrome c oxidase assembly factor in Saccharomyces cerevisiae

The twin-Cx(9)C motif protein Pet191 is essential for cytochrome c oxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx(9)C motif class of proteins in being independent of the Mia40 pathway.     

Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial cytochrome c oxidase and adenosine triphosphate synthase complex.

The interaction between cytochrome c oxidase complex and adenosine triphosphate synthase (F1F0) complex in the purified, dispersed state and embedded in phospholipid vesicles was studied by differential scanning calorimetry and by spin-label electron paramagnetic resonance. The detergent-dispersed cytochrome oxidase and F1F0 complexes undergo endothermic thermodenaturation. However, when these complexes are embedded in phospholipid vesicles, they undergo exothermic thermodenaturation. The energy released is believed to result from the collapse of a strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by the removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both cytochrome oxidase complex and F1F0 was smaller than that of a mixture of protein-phospholipid vesicles formed from each individual electron transfer complex. This suggests specific interaction between cytochrome oxidase complex and F1F0 in the membrane. Further evidence for interaction between these two complexes is provided by saturation transfer EPR studies in which the rotational correlation time of spin-labeled cytochrome oxidase increases significantly when the complex is mixed with F1F0 prior to being embedded in phospholipid vesicles. From these results, it is concluded that at least a part of cytochrome oxidase and a part of F1F0 form a supermacromolecular complex in the inner mitochondrial membrane. No such supermacromolecular complex is detected between F1F0 and ubiquinol--cytochrome c reductase.     

Further insights into the assembly of the yeast cytochrome bc1 complex based on analysis of single and double deletion mutants lacking supernumerary subunits and cytochrome b.

The cytochrome bc1 complex of the yeast Saccharomyces cerevisiae is composed of 10 different subunits that are assembled as a symmetrical dimer in the inner mitochondrial membrane. Three of the subunits contain redox centers and participate in catalysis, whereas little is known about the function of the seven supernumerary subunits. To gain further insight into the function of the supernumerary subunits in the assembly process, we have examined the subunit composition of mitochondrial membranes isolated from yeast mutants in which the genes for supernumerary subunits and cytochrome b were deleted and from yeast mutants containing double deletions of supernumerary subunits. Deletion of any one of the genes encoding cytochrome b, subunit 7 or subunit 8 caused the loss of the other two subunits. This is consistent with the crystal structure of the cytochrome bc1 complex that shows that these three subunits comprise its core, around which the remaining subunits are assembled. Absence of the cytochrome b/subunit 7/subunit 8 core led to the loss of subunit 6, whereas cytochrome c1, iron-sulfur protein, core protein 1, core protein 2 and subunit 9 were still assembled in the membrane, although in reduced amounts. Parallel changes in the amounts of core protein 1 and core protein 2 in the mitochondrial membranes of all of the deletion mutants suggest that these can be assembled as a subcomplex in the mitochondrial membrane, independent of the presence of any other subunits. Likewise, evidence of interactions between subunit 6, subunit 9 and cytochrome c1 suggests that a subcomplex between these two supernumerary subunits and the cytochrome might exist.     

Copper chaperones for cytochrome c oxidase and human disease

Biological processes in living cells are compartmentalized between lipid membranes. Integral membrane proteins often confer specific functions to these compartments and as such have a critical role in cellular metabolism and function. Cytochrome c oxidase is a macromolecular metalloprotein complex essential for the respiratory function of the cell. Elucidating the mechanisms of assembly of cytochrome c oxidase within the inner mitochondrial membrane represents a unique challenge for understanding metalloprotein biosynthesis. Elegant genetic experiments in yeast have defined several proteins required for copper delivery to cytochrome c oxidase. While the precise role of each of these proteins in copper incorporation remains unclear, recent studies have revealed that inherited mutations in two of these proteins can result in severe pathology in human infants in association with cytochrome c oxidase deficiency. Characterization of the molecular pathogenesis of these disorders offers new insights into the mechanisms of cellular copper metabolism and the role of these cytochrome c oxidase copper chaperones in human disease.     

The yeast Aac2 protein exists in physical association with the cytochrome bc1-COX supercomplex and the TIM23 machinery

The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc(1))-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc(1)-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc(1)-COX supercomplex is perturbed, whereby a decrease in the III(2)-IV(2) assembly state relative to the III(2)-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc(1)-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc(1)-COX-TIM23 supercomplex are also discussed.     

Inhibitor-complexed structures of the cytochrome bc1 from the photosynthetic bacterium Rhodobacter sphaeroides

The cytochrome bc(1) complex (bc(1)) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc(1) complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc(1)), stabilized with the quinol oxidation (Q(P)) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q(N)) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c(1) subunit and a number of lipid and detergent molecules. Structural differences between Rsbc(1) and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc(1) complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.     

Transport of proteins to the mitochondrial intermembrane space: the ''sorting'' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane.

The presequence of yeast cytochrome c1 (an inner membrane protein protruding into the intermembrane space) contains a matrix-targeting domain and an intramitochondrial sorting domain. This presequence transports attached subunit IV of cytochrome c oxidase into the intermembrane space (van Loon et al. (1987) EMBO J., 6, 2433-2439). In order to determine how this fusion protein reaches the intermembrane space, we studied the kinetics of its import into isolated mitochondria or mitoplasts and its accumulation in the various submitochondrial compartments. The imported, uncleaved fusion precursor and a cleavage intermediate were bound to the inner membrane and were always exposed to the intermembrane space; they were never found at the matrix side of the inner membrane. In contrast, analogous import experiments with the authentic subunit IV precursor, or the precursor of the iron-sulphur protein of the cytochrome bc1 complex also an inner membrane protein exposed to the intermembrane space), readily showed that these precursors were initially transported across both mitochondrial membranes. We conclude that the intramitochondrial sorting domain within the cytochrome c1 presequence prevents transport of attached proteins across the inner, but not the outer membrane: it is a stop-transfer sequence for the inner membrane. Since the presequence of the iron-sulphur protein lacks such 'stop-transfer' domain, it acts by a different mechanism.     

Purification of a cytochrome bc-aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum. Identification of a fourth subunity of cytochrome aa3 oxidase and mutational analysis of diheme cytochrome c1

The aerobic respiratory chain of the Gram-positive Corynebacterium glutamicum involves a bc(1) complex with a diheme cytochrome c(1) and a cytochrome aa(3) oxidase but no additional c-type cytochromes. Here we show that the two enzymes form a supercomplex, because affinity chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochrome aa(3) always resulted in the copurification of the subunits of the bc(1) complex (QcrA, QcrB, QcrC) and the aa(3) complex (CtaD, CtaC, CtaE). The isolated bc(1)-aa(3) supercomplexes had quinol oxidase activity, indicating functional electron transfer between cytochrome c(1) and the Cu(A) center of cytochrome aa(3). Besides the known bc(1) and aa(3) subunits, few additional proteins were copurified, one of which (CtaF) was identified as a fourth subunit of cytochrome aa(3). If either of the two CXXCH motifs for covalent heme attachment in cytochrome c(1) was changed to SXXSH, the resulting mutants showed severe growth defects, had no detectable c-type cytochrome, and their cytochrome b level was strongly reduced. This indicates that the attachment of both heme groups to apo-cytochrome c(1) is not only required for the activity but also for the assembly and/or stability of the bc(1) complex.     

Cytochrome bc(1) regulates the mitochondrial permeability transition by two distinct pathways

The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the mitochondrial inner membrane that plays a crucial role in cell death. Here we show that cytochrome bc(1) regulates the MPT in isolated rat liver mitochondria and in CEM and HL60 cells by two independent pathways. Glutathione depletion activated the MPT via increased production of reactive oxygen species (ROS) generated by cytochrome bc(1). The ROS producing mechanism in cytochrome bc(1) involves movement of the "Rieske" iron-sulfur protein subunit of the enzyme complex, because inhibition of cytochrome bc(1) by pharmacologically blocking iron-sulfur protein movement completely abolished ROS production, MPT activation, and cell death. The classical inhibitor of the MPT, cyclosporine A, had no protective effect against MPT activation. In contrast, the calcium-activated, cyclosporine A-regulated MPT in rat liver mitochondria was also blocked with inhibitors of cytochrome bc(1). These results indicate that electron flux through cytochrome bc(1) regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial ROS and the other regulated and activated by calcium.     

Synthesis and intracellular transport of cytochrome oxidase subunit IV and ADP/ATP translocator protein in intact hepatoma cells

The biosynthesis of two mitochondrial membrane proteins - subunit IV of cytochrome oxidase and ADP/ATP translocator protein was studied in intact ascites hepatoma cells. Using pulse-chase labeling and rapid cell fractionation it was possible to identify the precursoric forms of these inner mitochondrial membrane proteins. It was found that the subunit IV of cytochrome oxidase is synthesized in the cytoplasm of mammalian cells in the form of a larger precursor while ADP/ATP translocator protein is synthesized in the form that is electrophoretically undistinguishable from the mature membrane integrated form.     

Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites

Atovaquone represents a class of antimicrobial agents with a broad-spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria-containing parasites. To that end, we derived nine independent atovaquone-resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone-mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance-conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side-chains may determine atovaquone-binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.