Mass Spectrometric Analysis of Purine Intermediary Metabolism Indicates Cyanide Induces Purine Catabolism in Rabbits.

Purines are the building blocks of DNA/RNA, energy substrates, and cofactors. Purine metabolites, including ATP, GTP, NADH, and coenzyme A, are essential molecules in diverse biological processes such as energy metabolism, signal transduction, and enzyme activity. When purine levels increase, excess purines are either recycled to synthesize purine metabolites or catabolized to the end product uric acid. Purine catabolism increases during states of low oxygen tension (hypoxia and ischemia), but this metabolic pathway is incompletely understood in the context of histotoxic hypoxia (i.e., inhibition of oxygen utilization despite normal oxygen tension). In rabbits exposed to cyanide-a classical histotoxic hypoxia agent-we demonstrated significant increases in several concordant metabolites in the purine catabolic pathway (including plasma levels of uric acid, xanthosine, xanthine, hypoxanthine, and inosine) via mass spectrometry-based metabolite profiling. Pharmacological inhibition of the purine catabolic pathway with oxypurinol mitigated the deleterious effects of cyanide on skeletal muscle cytochrome c oxidase redox state, measured by non-invasive diffuse optical spectroscopy. Finally, plasma uric acid levels correlated strongly with those of lactic acid, an established clinical biomarker of cyanide exposure, in addition to a tissue biomarker of cyanide exposure (skeletal muscle cytochrome c oxidase redox state). Cumulatively, these findings not only shed light on the in vivo role(s) of cyanide but also have implications in the field of medical countermeasure (MCM) development.

How Azide Ion/Hydrazoic Acid Passes Through Biological Membranes: An Experimental and Computational Study.

Hydrazoic acid (HN3) and its deprotonated form azide ion (N3-) (AHA) are toxic because they inhibit the cytochrome c oxidase complex IV (CoX IV) embedded in the inner mitochondrial membrane that forms part of the enzyme complexes involved in cellular respiration. Critical to its toxicity is the inhibition of CoX IV in the central nervous system and cardiovascular system. Hydrazoic acid is an ionizable species and its affinity for membranes, and the associated permeabilities, depend on the pH values of aqueous media on both sides of the membranes. In this article, we address the permeability of AHA through the biological membrane. In order to understand the affinity of the membrane for the neutral and ionized form of azide, we measured the octanol/water partition coefficients at pH values of 2.0 and 8.0, which are 2.01 and 0.00034, respectively. Using a Parallel Artificial Membrane Permeability Assay (PAMPA) experiment, we measured the effective permeability through the membrane, which is logPe - 4.97 and - 5.26 for pH values of 7.4 and pH 8.0, respectively. Experimental permeability was used to validate theoretical permeability, which was estimated by numerically solving a Smoluchowski equation for AHA diffusion through the membrane. We demonstrated that the rate of permeation through the cell membrane of 8.46·104 s-1 is much higher than the rate of the chemical step of CoX IV inhibition by azide of 200 s-1. The results of this study show that transport through the membrane does not represent the rate-limiting step and therefore does not control the rate of CoX IV inhibition in the mitochondria. However, the observed dynamics of azide poisoning is controlled by circulatory transport that takes place on a time scale of minutes.

Evidence that the catalytic mechanism of heme a synthase involves the formation of a carbocation stabilized by a conserved glutamate.

In eukaryotes and many aerobic prokaryotes, the final step of aerobic respiration is catalyzed by an aa3-type cytochrome c oxidase, which requires a modified heme cofactor, heme a. The conversion of heme b, the prototypical cellular heme, to heme o and ultimately to heme a requires two modifications, the latter of which is conversion of a methyl group to an aldehyde, catalyzed by heme a synthase (HAS). The N- and C-terminal halves of HAS share homology, and each half contains a heme-binding site. Previous reports indicate that the C-terminal site is occupied by a heme b cofactor. The N-terminal site may function as the substrate (heme o) binding site, although this has not been confirmed experimentally. Here, we assess the role of conserved residues from the N- and C-terminal heme-binding sites in HAS from prokaryotic (Shewanella oneidensis) and eukaryotic (Saccharomyces cerevisiae) species - SoHAS/CtaA and ScHAS/Cox15, respectively. A glutamate within the N-terminal site is found to be critical for activity in both types of HAS, consistent with the hypothesis that a carbocation forms transiently during catalysis. In contrast, the residue occupying the analogous C-terminal position is dispensable for enzyme activity. In SoHAS, the C-terminal heme ligands are critical for stability, while in ScHAS, substitutions in either heme-binding site have little effect on global structure. In both species, in vivo accumulation of heme o requires the presence of an inactive HAS variant, highlighting a potential regulatory role for HAS in heme o biosynthesis.

Retinoic acid exerts sexually dimorphic effects on muscle energy metabolism and function.

The retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction that converts retinol into retinoic acid (RA), an autacoid that regulates energy balance and reduces adiposity. Skeletal muscle contributes to preventing adiposity, by consuming nearly half the energy of a typical human. We report sexually dimorphic differences in energy metabolism and muscle function in Rdh10+/- mice. Relative to wild-type (WT) controls, Rdh10+/- males fed a high-fat diet decrease reliance on fatty-acid oxidation and experience glucose intolerance and insulin resistance. Running endurance decreases 40%. Rdh10+/- females fed this diet increase fatty acid oxidation and experience neither glucose intolerance nor insulin resistance. Running endurance increases 220%. We therefore assessed RA function in the mixed-fiber type gastrocnemius muscles (GM), which contribute to running, rather than standing, and are similar to human GM. RA levels in Rdh10+/- male GM decrease 38% relative to WT. Rdh10+/- male GM increase expression of Myog and reduce Eif6 mRNAs, which reduce and enhance running endurance, respectively. Cox5A, complex IV activity, and ATP decrease. Increased centralized nuclei reveal existence of muscle malady and/or repair in GM fibers. Comparatively, RA in Rdh10+/- female GM decreases by less than half the male decrease, from a more modest decrease in Rdh10 and an increase in the estrogen-induced retinol dehydrogenase Dhrs9. Myog mRNA decreases. Cox5A, complex IV activity, and ATP increase. Centralized GM nuclei do not increase. We conclude that Rdh10/RA affects whole body energy use and insulin resistance partially through sexual dimorphic effects on skeletal muscle gene expression, structure, and mitochondria activity.

Critical roles of the CuB site in efficient proton pumping as revealed by crystal structures of mammalian cytochrome c oxidase catalytic intermediates.

Mammalian cytochrome c oxidase (CcO) reduces O2 to water in a bimetallic site including Fea3 and CuB giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by single-electron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a proton-conducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at ∼1.8 Å resolution of fully reduced CcO crystals treated with O2 for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained ∼45% of the O-form structure, ∼45% of the E-form structure, and ∼20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O- and E-forms exhibit an unusually long CuB2+-OH- distance and CuB1+-H2O structure keeping Fea33+-OH- state, respectively, suggesting that the O- and E-forms have high electron affinities that cause the O→E and E→R transitions to be essentially irreversible and thus enable tightly coupled proton pumping. The water channel of the H-pathway is closed in the O- and E-forms and partially open in the R-form. These structures, together with those of the recently reported P- and F-forms, indicate that closure of the H-pathway water channel avoids back-leaking of protons for facilitating the effective proton pumping.

X-ray structures of catalytic intermediates of cytochrome c oxidase provide insights into its O2 activation and unidirectional proton-pump mechanisms.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fe a32+ and CuB1+, and suggests that a peroxide-bound state (Fe a33+-O--O--CuB2+) rather than an O2-bound state (Fe a32+-O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fe a32+-O2, whereas Fe a33+-O--O--CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Šresolution. A 1.70 ŠFe-O distance of the ferryl center could best be described as Fe a34+ = O2-, not as Fe a34+-OH- The distance suggests an ∼800-cm-1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fe a33+-O--O--CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Hypoxia-inducible gene domain 1 proteins in yeast mitochondria protect against proton leak through complex IV.

Hypoxia-inducible gene domain 1 (HIGD1) proteins are small integral membrane proteins, conserved from bacteria to humans, that associate with oxidative phosphorylation supercomplexes. Using yeast as a model organism, we have shown previously that its two HIGD1 proteins, Rcf1 and Rcf2, are required for the generation and maintenance of a normal membrane potential (ΔΨ) across the inner mitochondrial membrane (IMM). We postulated that the lower ΔΨ observed in the absence of the HIGD1 proteins may be due to decreased proton pumping by complex IV (CIV) or enhanced leak of protons across the IMM. Here we measured the ΔΨ generated by complex III (CIII) to discriminate between these possibilities. First, we found that the decreased ΔΨ observed in the absence of the HIGD1 proteins cannot be due to decreased proton pumping by CIV because CIII, operating alone, also exhibited a decreased ΔΨ when HIGD1 proteins were absent. Because CIII can neither lower its pumping stoichiometry nor transfer protons completely across the IMM, this result indicates that HIGD1 protein ablation enhances proton leak across the IMM. Second, we demonstrate that this proton leak occurs through CIV because ΔΨ generation by CIII is restored when CIV is removed from the cell. Third, the proton leak appeared to take place through an inactive population of CIV that accumulates when HIGD1 proteins are absent. We conclude that HIGD1 proteins in yeast prevent CIV inactivation, likely by preventing the loss of lipids bound within the Cox3 protein of CIV.

Yeast mitochondrial protein Pet111p binds directly to two distinct targets in COX2 mRNA, suggesting a mechanism of translational activation.

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in vitro analyses to characterize the interactions of one of these translational activators, the pentatricopeptide repeat protein Pet111p, with its presumed target, COX2 mRNA, which encodes subunit II of cytochrome c oxidase. Using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation analysis, we found that Pet111p binds directly and specifically to a 5'-end proximal region of the COX2 transcript. Further, we applied in vitro RNase footprinting and mapped two binding targets of the protein, of which one is located in the 5'-untranslated leader and the other is within the coding sequence. Combined with the available genetic data, these results suggest a plausible mechanism of translational activation, in which binding of Pet111p may prevent inhibitory secondary structures from forming in the translation initiation region, thus rendering the mRNA available for interaction with the ribosome.

Cox15 interacts with the cytochrome bc1 dimer within respiratory supercomplexes as well as in the absence of cytochrome c oxidase.

The heme a molecule is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome c oxidase. Heme a is synthesized from heme o by a multi-spanning inner membrane protein, heme a synthase (Cox15 in the yeast Saccharomyces cerevisiae). The insertion of heme a is critical for cytochrome c oxidase function and assembly, but this process has not been fully elucidated. To improve our understanding of heme a insertion into cytochrome c oxidase, here we investigated the protein-protein interactions that involve Cox15 in S. cerevisiae In addition to observing Cox15 in homooligomeric complexes, we found that a portion of Cox15 also associates with the mitochondrial respiratory supercomplexes. When supercomplex formation was abolished, as in the case of stalled cytochrome bc1 or cytochrome c oxidase assembly, Cox15 maintained an interaction with select proteins from both respiratory complexes. In the case of stalled cytochrome bc1 assembly, Cox15 interacted with the late-assembling cytochrome c oxidase subunit, Cox13. When cytochrome c oxidase assembly was stalled, Cox15 unexpectedly maintained its interaction with the cytochrome bc1 protein, Cor1. Our results indicate that Cox15 and Cor1 continue to interact in the cytochrome bc1 dimer even in the absence of supercomplexes or when the supercomplexes are destabilized. These findings reveal that Cox15 not only associates with respiratory supercomplexes, but also interacts with the cytochrome bc1 dimer even in the absence of cytochrome c oxidase.

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules.

Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix to the intermembrane space. In the yeast Saccharomyces cerevisiae, COX is composed of 11-13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates. Analysis of radiolabeled Cox2p obtained in pulldown assays by native gel electrophoresis revealed the existence of several assembly intermediates, the largest of which had an estimated mass of 450-550 kDa. None of the other known subunits of COX were present in these Cox2p intermediates. This was also true for the several ancillary factors having still undefined functions in COX assembly. In agreement with earlier evidence, Cox18p and Cox20p, previously shown to be involved in processing and in membrane insertion of the Cox2p precursor, were found to be associated with the two largest Cox2p intermediates. A small fraction of the Cox2p module contained Sco1p and Coa6p, which have been implicated in metalation of the binuclear copper site on this subunit. Our results indicate that following its insertion into the mitochondrial inner membrane, Cox2p assembles as a stand-alone protein with the compositionally more complex Cox1p and Cox3p modules.

X-ray structural analyses of azide-bound cytochrome c oxidases reveal that the H-pathway is critically important for the proton-pumping activity.

Cytochrome c oxidase (CcO) is the terminal oxidase of cellular respiration, reducing O2 to water and pumping protons. X-ray structural features have suggested that CcO pumps protons via a mechanism involving electrostatic repulsions between pumping protons in the hydrogen-bond network of a proton-conducting pathway (the H-pathway) and net positive charges created upon oxidation of an iron site, heme a (Fe a2+), for reduction of O2 at another iron site, heme a3 (Fe a32+). The protons for pumping are transferred to the hydrogen-bond network from the N-side via the water channel of the H-pathway. Back-leakage of protons to the N-side is thought to be blocked by closure of the water channel. To experimentally test this, we examined X-ray structures of the azide-bound, oxidized bovine CcO and found that an azide derivative (N3--Fe a33+, CuB2+-N3-) induces a translational movement of the heme a3 plane. This was accompanied by opening of the water channel, revealing that Fe a3 and the H-pathway are tightly coupled. The channel opening in the oxidized state is likely to induce back-leakage of pumping protons, which lowers the proton level in the hydrogen-bond network during enzymatic turnover. The proton level decrease weakens the electron affinity of Fe a , if Fe a electrostatically interacts with protons in the hydrogen-bond network. The previously reported azide-induced redox-potential decrease in Fe a supports existence of the electrostatic interaction. In summary, our results indicate that the H-pathway is critical for CcO's proton-pumping function.

Ethanolamine ameliorates mitochondrial dysfunction in cardiolipin-deficient yeast cells.

Cardiolipin (CL) is a signature phospholipid of the mitochondria required for the formation of mitochondrial respiratory chain (MRC) supercomplexes. The destabilization of MRC supercomplexes is the proximal cause of the pathology associated with the depletion of CL in patients with Barth syndrome. Thus, promoting supercomplex formation could ameliorate mitochondrial dysfunction associated with CL depletion. However, to date, physiologically relevant small-molecule regulators of supercomplex formation have not been identified. Here, we report that ethanolamine (Etn) supplementation rescues the MRC defects by promoting supercomplex assembly in a yeast model of Barth syndrome. We discovered this novel role of Etn while testing the hypothesis that elevating mitochondrial phosphatidylethanolamine (PE), a phospholipid suggested to overlap in function with CL, could compensate for CL deficiency. We found that the Etn supplementation rescues the respiratory growth of CL-deficient Saccharomyces cerevisiae cells in a dose-dependent manner but independently of its incorporation into PE. The rescue was specifically dependent on Etn but not choline or serine, the other phospholipid precursors. Etn improved mitochondrial function by restoring the expression of MRC proteins and promoting supercomplex assembly in CL-deficient cells. Consistent with this mechanism, overexpression of Cox4, the MRC complex IV subunit, was sufficient to promote supercomplex formation in CL-deficient cells. Taken together, our work identifies a novel role of a ubiquitous metabolite, Etn, in attenuating mitochondrial dysfunction caused by CL deficiency.

The cellular stress proteins CHCHD10 and MNRR1 (CHCHD2): Partners in mitochondrial and nuclear function and dysfunction.

Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) and CHCHD2 (MNRR1) are homologous proteins with 58% sequence identity and belong to the twin CX9C family of proteins that mediate cellular stress responses. Despite the identification of several neurodegeneration-associated mutations in the CHCHD10 gene, few studies have assessed its physiological role. Here, we investigated CHCHD10's function as a regulator of oxidative phosphorylation in the mitochondria and the nucleus. We show that CHCHD10 copurifies with cytochrome c oxidase (COX) and up-regulates COX activity by serving as a scaffolding protein required for MNRR1 phosphorylation, mediated by ARG (ABL proto-oncogene 2, nonreceptor tyrosine kinase (ABL2)). The CHCHD10 gene was maximally transcribed in cultured cells at 8% oxygen, unlike MNRR1, which was maximally expressed at 4%, suggesting a fine-tuned oxygen-sensing system that adapts to the varying oxygen concentrations in the human body under physiological conditions. We show that nuclear CHCHD10 protein down-regulates the expression of genes harboring the oxygen-responsive element (ORE) in their promoters by interacting with and augmenting the activity of the largely uncharacterized transcriptional repressor CXXC finger protein 5 (CXXC5). We further show that two genetic CHCHD10 disease variants, G66V and P80L, in the mitochondria exhibit faulty interactions with MNRR1 and COX, reducing respiration and increasing reactive oxygen species (ROS), and in the nucleus abrogating transcriptional repression of ORE-containing genes. Our results reveal that CHCHD10 positively regulates mitochondrial respiration and contributes to transcriptional repression of ORE-containing genes in the nucleus, and that genetic CHCHD10 variants are impaired in these activities.

Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase.

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.

Filling the mitochondrial copper pool.

A host of critical metalloproteins reside in mitochondria, where metallation occurs within the organelle after protein import. Although the pathways by which proteins are imported into the mitochondria are well known, the mechanisms by which their metal partners are imported are more obscure. A new study by Boulet et al. demonstrates that the mammalian SLC25A3 inner membrane transporter, previously known as a phosphate carrier, is also a functional Cu(I) importer, clarifying the source of mitochondrial copper and raising new questions about cellular copper homeostasis.

Building the CuA site of cytochrome c oxidase: A complicated, redox-dependent process driven by a surprisingly large complement of accessory proteins.

Cytochrome c oxidase (COX) was initially purified more than 70 years ago. A tremendous amount of insight into its structure and function has since been gleaned from biochemical, biophysical, genetic, and molecular studies. As a result, we now appreciate that COX relies on its redox-active metal centers (heme a and a3, CuA and CuB) to reduce oxygen and pump protons in a reaction essential for most eukaryotic life. Questions persist, however, about how individual structural subunits are assembled into a functional holoenzyme. Here, we focus on what is known and what remains to be learned about the accessory proteins that facilitate CuA site maturation.

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis.

Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.

Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase.

Mitochondrial cytochrome oxidase (COX) catalyzes the last step in the respiratory pathway. In the yeast Saccharomyces cerevisiae, this inner membrane complex is composed of 11 protein subunits. Expression of COX is assisted by some two dozen ancillary proteins that intercede at different stages of the assembly pathway. One such protein, Cox16p, encoded by COX16, was shown to be essential for the activity and assembly of COX. The function of Cox16p, however, has not been determined. We present evidence that Cox16p is present in Cox1p assembly intermediates and in COX. This is based on the finding that Cox16p, tagged with a dual polyhistidine and protein C tag, co-immunopurified with Cox1p assembly intermediates. The pulldown assays also indicated the presence of Cox16p in mature COX and in supercomplexes consisting of COX and the bc1 complex. From the Western signal strengths, Cox16p appears to be substoichiometric with Cox1p and Cox4p, which could indicate that Cox16p is only present in a fraction of COX. In conclusion, our results indicate that Cox16p is a constituent of several Cox1p assembly intermediates and of COX.

The Cox1 C-terminal domain is a central regulator of cytochrome c oxidase biogenesis in yeast mitochondria.

Cytochrome c oxidase (CcO) is the last electron acceptor in the respiratory chain. The CcO core is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if CcO assembly is blocked, Cox1 synthesis decreases. Mss51 activates translation of COX1 mRNA and interacts with Cox1 protein in high-molecular-weight complexes (COA complexes) to form the Cox1 intermediary assembly module. Thus, Mss51 coordinates both Cox1 synthesis and assembly. We previously reported that the last 15 residues of the Cox1 C terminus regulate Cox1 synthesis by modulating an interaction of Mss51 with Cox14, another component of the COA complexes. Here, using site-directed mutagenesis of the mitochondrial COX1 gene from Saccharomyces cerevisiae, we demonstrate that mutations P521A/P522A and V524E disrupt the regulatory role of the Cox1 C terminus. These mutations, as well as C terminus deletion (Cox1ΔC15), reduced binding of Mss51 and Cox14 to COA complexes. Mss51 was enriched in a translationally active form that maintains full Cox1 synthesis even if CcO assembly is blocked in these mutants. Moreover, Cox1ΔC15, but not Cox1-P521A/P522A and Cox1-V524E, promoted formation of aberrant supercomplexes in CcO assembly mutants lacking Cox2 or Cox4 subunits. The aberrant supercomplex formation depended on the presence of cytochrome b and Cox3, supporting the idea that supercomplex assembly factors associate with Cox3 and demonstrating that supercomplexes can be formed even if CcO is inactive and not fully assembled. Our results indicate that the Cox1 C-terminal end is a key regulator of CcO biogenesis and that it is important for supercomplex formation/stability.

Nanomolar nitric oxide concentrations quickly and reversibly modulate astrocytic energy metabolism.

Nitric oxide (NO) is an intercellular messenger involved in multiple bodily functions. Prolonged NO exposure irreversibly inhibits respiration by covalent modification of mitochondrial cytochrome oxidase, a phenomenon of pathological relevance. However, the speed and potency of NO's metabolic effects at physiological concentrations are incompletely characterized. To this end, we set out to investigate the metabolic effects of NO in cultured astrocytes from mice by taking advantage of the high spatiotemporal resolution afforded by genetically encoded Förster resonance energy transfer (FRET) nanosensors. NO exposure resulted in immediate and reversible intracellular glucose depletion and lactate accumulation. Consistent with cytochrome oxidase involvement, the glycolytic effect was enhanced at a low oxygen level and became irreversible at a high NO concentration or after prolonged exposure. Measurements of both glycolytic rate and mitochondrial pyruvate consumption revealed significant effects even at nanomolar NO concentrations. We conclude that NO can modulate astrocytic energy metabolism in the short term, reversibly, and at concentrations known to be released by endothelial cells under physiological conditions. These findings suggest that NO modulates the size of the astrocytic lactate reservoir involved in neuronal fueling and signaling.