hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Copper Starvation-inducible Protein for Cytochrome Oxidase Biogenesis in Bradyrhizobium japonicum

Microarray analysis of Bradyrhizobium japonicum grown under copper limitation uncovered five genes named pcuABCDE, which are co-transcribed and co-regulated as an operon. The predicted gene products are periplasmic proteins (PcuA, PcuC, and PcuD), a TonB-dependent outer membrane receptor (PcuB), and a cytoplasmic membrane-integral protein (PcuE). Homologs of PcuC and PcuE had been discovered in other bacteria, namely PCu_AC and YcnJ, where they play a role in cytochrome oxidase biogenesis and copper transport, respectively. Deletion of the pcuABCDE operon led to a pleiotropic phenotype, including defects in the aa₃-type cytochrome oxidase, symbiotic nitrogen fixation, and anoxic nitrate respiration. Complementation analyses revealed that, under our assay conditions, the tested functions depended only on the pcuC gene and not on pcuA, pcuB, pcuD, or pcuE. The B. japonicum genome harbors a second pcuC-like gene (blr7088), which, however, did not functionally replace the mutated pcuC. The PcuC protein was overexpressed in Escherichia coli, purified to homogeneity, and shown to bind Cu(I) with high affinity in a 1:1 stoichiometry. The replacement of His⁷⁹, Met⁹⁰, His¹¹³, and Met¹¹⁵ by alanine perturbed copper binding. This corroborates the previously purported role of this protein as a periplasmic copper chaperone for the formation of the Cu_A center on the aa₃-type cytochrome oxidase. In addition, we provide evidence that PcuC and the copper chaperone ScoI are important for the symbiotically essential, Cu_A-free cbb₃-type cytochrome oxidase specifically in endosymbiotic bacteroids of soybean root nodules, which could explain the symbiosis-defective phenotype of the pcuC and scoI mutants.     

The Protein Effect in the Structure of Two Ferryl-Oxo Intermediates at the Same Oxidation Level in the Heme Copper Binuclear Center of Cytochrome c Oxidase

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O₂ activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O₂ with the mixed valence form (Cu_A²⁺, heme a³⁺, heme a₃²⁺-Cu_B¹⁺) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm⁻¹ at the peroxy oxidation level (P_M). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the P_M intermediate is also formed in the reaction of Y167F with O₂. These results suggest that in the fully reduced enzyme, the proton pumping ν_Fe(IV)=O = 804 cm⁻¹ to ν_Fe(IV)=O = 790 cm⁻¹ transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a₃ but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a₃. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a₃-Asp-399-H₂O site and, thus, to the exit/output proton channel (H₂O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a₃ controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.     

Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

Cytochrome aa₃-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O₂ to 2H₂O. Cytochrome aa₃-600 is of interest because it is a very close homologue of the cytochrome bo₃ ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo₃ has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa₃-600. Cytochrome aa₃-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa₃-600 in comparison with the ubi-semiquinone in cytochrome bo₃. Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa₃-600 in comparison with ubiquinone in cytochrome bo₃. In addition, the electronic structure of the semiquinone cyt aa₃-600 is more shifted toward the anionic form from the neutral state in cyt bo₃.     

Selective Oma1 protease-mediated proteolysis of Cox1 subunit of cytochrome oxidase in assembly mutants

Stalled biogenesis of the mitochondrial cytochrome c oxidase (CcO) complex results in degradation of subunits containing redox cofactors. The conserved Oma1 metalloproteinase mediates facile Cox1 degradation in cells lacking the Coa2 assembly factor, but not in a series of other mutants stalled in CcO maturation. Oma1 is activated in coa2Δ cells, but the selective Cox1 degradation does not arise merely from its activation. Oma1 is also active in cells with dysfunctional mitochondria and cox11Δ cells impaired in CcO maturation, but this activation does not result in Oma1-mediated Cox1 degradation. The facile and selective degradation of Cox1 in coa2Δ cells, relative to other CcO assembly mutants, is likely due to impaired hemylation and subsequent misfolding of the subunit. Specific Cox1 proteolysis in coa2Δ cells arises from a combination of Oma1 activation and a susceptible conformation of Cox1.     

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

The Atp9p ring is one of several assembly modules of yeast mitochondrial ATP synthase. The ring, composed of 10 copies of Atp9p, is part of the rotor that couples proton translocation to synthesis or hydrolysis of ATP. We present evidence that before its assembly with other ATP synthase modules, most of Atp9p is present in at least three complexes with masses of 200–400 kDa that co-immunopurify with Cox6p. Pulse-labeling analysis disclosed a time-dependent reduction of radiolabeled Atp9p in the complexes and an increase of Atp9p in the ring form of wild type yeast and of mss51, pet111, and pet494 mutants lacking Cox1p, Cox2p, and Cox3p, respectively. Ring formation was not significantly different from wild type in an mss51 or atp10 mutant. The atp10 mutation blocks the interaction of the Atp9p ring with other modules of the ATP synthase. In contrast, ring formation was reduced in a cox6 mutant, consistent with a role of Cox6p in oligomerization of Atp9p. Cox6p involvement in ATP synthase assembly is also supported by studies showing that ring formation in cells adapting from fermentative to aerobic growth was less efficient in mitochondria of the cox6 mutant than the parental respiratory-competent strain or a cox4 mutant. We speculate that the constitutive and Cox6p-independent rate of Atp9p oligomerization may be sufficient to produce the level of ATP synthase needed for maintaining a membrane potential but limiting for optimal oxidative phosphorylation.     

The Carboxyl-terminal End of Cox1 Is Required for Feedback Assembly Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria

Synthesis of the largest cytochrome c oxidase (CcO) subunit, Cox1, on yeast mitochondrial ribosomes is coupled to assembly of CcO. The translational activator Mss51 is sequestered in early assembly intermediate complexes by an interaction with Cox14 that depends on the presence of newly synthesized Cox1. If CcO assembly is prevented, the level of Mss51 available for translational activation is reduced. We deleted the C-terminal 11 or 15 residues of Cox1 by site-directed mutagenesis of mtDNA. Although these deletions did not prevent respiratory growth of yeast, they eliminated the assembly-feedback control of Cox1 synthesis. Furthermore, these deletions reduced the strength of the Mss51-Cox14 interaction as detected by co-immunoprecipitation, confirming the importance of the Cox1 C-terminal residues for Mss51 sequestration. We surveyed a panel of mutations that block CcO assembly for the strength of their effect on Cox1 synthesis, both by pulse labeling and expression of the ARG8_m reporter fused to COX1. Deletion of the nuclear gene encoding Cox6, one of the first subunits to be added to assembling CcO, caused the most severe reduction in Cox1 synthesis. Deletion of the C-terminal 15 amino acids of Cox1 increased Cox1 synthesis in the presence of each of these mutations, except pet54. Our data suggest a novel activity of Pet54 required for normal synthesis of Cox1 that is independent of the Cox1 C-terminal end.     

Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.     

Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction

In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.     

Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion

Mutations in FBXL4 (F-Box and Leucine rich repeat protein 4), a nuclear-encoded mitochondrial protein with an unknown function, cause mitochondrial DNA depletion syndrome. We report two siblings, from consanguineous parents, harbouring a previously uncharacterized homozygous variant in FBXL4 (c.1750 T > C; p.Cys584Arg). Both patients presented with encephalomyopathy, lactic acidosis and cardiac hypertrophy, which are reported features of FBXL4 impairment. Remarkably, dichloroacetate (DCA) administration to the younger sibling improved metabolic acidosis and reversed cardiac hypertrophy. Characterization of FBXL4 patient fibroblasts revealed severe bioenergetic defects, mtDNA depletion, fragmentation of mitochondrial networks, and abnormalities in mtDNA nucleoids. These phenotypes, observed with other pathogenic FBXL4 variants, confirm the pathogenicity of the p.Cys584Arg variant. Although treating FBXL4 fibroblasts with DCA improved extracellular acidification, in line with reduced lactate levels in patients, DCA treatment did not improve any of the other mitochondrial functions. Nonetheless, we highlight DCA as a potentially effective drug for the management of elevated lactate and cardiomyopathy in patients with pathogenic FBXL4 variants. Finally, as the exact mechanism through which FBXL4 mutations lead to mtDNA depletion was unknown, we tested the hypothesis that FBXL4 promotes mitochondrial fusion. Using a photo-activatable GFP fusion assay, we found reduced mitochondrial fusion rates in cells harbouring a pathogenic FBXL4 variant. Meanwhile, overexpression of wildtype FBXL4, but not the p.Cys584Arg variant, promoted mitochondrial hyperfusion. Thus, we have uncovered a novel function for FBXL4 in promoting mitochondrial fusion, providing important mechanistic insights into the pathogenic mechanism underlying FBXL4 dysfunction.