Yeast Cytochrome Oxidase Research Articles

A human SCO2 mutation helps define the role of Sco1p in the cytochrome oxidase assembly pathway.

Deficiencies in cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, are most often caused by an inability to complete assembly of the enzyme. Pathogenic mutations in SCO2, which encodes a cytochrome oxidase assembly factor, were recently described in several cases of fatal infantile cardioencephalomyopathy. To determine the molecular etiology of these disorders, we describe the generation and characterization of the parallel mutations in the homologous yeast SCO1 gene. We show that the E155K yeast sco1 mutant is respiration-competent, whereas the S240F mutant is not. Interestingly, the S240F mutation allows partial but incorrect assembly of cytochrome oxidase, as judged by an altered cytochrome aa(3) peak. Immunoblot analysis reveals a specific absence of subunit 2 from the cytochrome oxidase in this mutant. Taken together, our data suggest that Sco1p provides copper to the Cu(A) site on subunit 2 at a step occurring late in the assembly pathway. This is the first instance of a yeast cytochrome oxidase assembly mutant that is partially assembled. The S240F mutant also represents a powerful new tool with which to elucidate further steps in the cytochrome oxidase assembly pathway.

Modular assembly of yeast mitochondrial ATP synthase and cytochrome oxidase

The respiratory pathway of mitochondria is composed of four electron transfer complexes and the ATP synthase. In this article, we review evidence from studies of Saccharomyces cerevisiae that both ATP synthase and cytochrome oxidase (COX) are assembled from independent modules that correspond to structurally and functionally identifiable components of each complex. Biogenesis of the respiratory chain requires a coordinate and balanced expression of gene products that become partner subunits of the same complex, but are encoded in the two physically separated genomes. Current evidence indicates that synthesis of two key mitochondrial encoded subunits of ATP synthase is regulated by the F1 module. Expression of COX1 that codes for a subunit of the COX catalytic core is also regulated by a mechanism that restricts synthesis of this subunit to the availability of a nuclear-encoded translational activator. The respiratory chain must maintain a fixed stoichiometry of the component enzyme complexes during cell growth. We propose that high-molecular-weight complexes composed of Cox6, a subunit of COX, and of the Atp9 subunit of ATP synthase play a key role in establishing the ratio of the two complexes during their assembly.

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.

Coi1 is a novel assembly factor of the yeast complex III-complex IV supercomplex.

The yeast bc1 complex (complex III) and cytochrome oxidase (complex IV) are mosaics of core subunits encoded by the mitochondrial genome and additional nuclear-encoded proteins imported from the cytosol. Both complexes build in the mitochondrial inner membrane various supramolecular assemblies. The formation of the individual complexes and their supercomplexes depends on the activity of dedicated assembly factors. We identified a so far uncharacterized mitochondrial protein (open reading frame YDR381C-A) as an important assembly factor for complex III, complex IV, and their supercomplexes. Therefore, we named this protein Cox interacting (Coi) 1. Deletion of COI1 results in decreased respiratory growth, reduced membrane potential, and hampered respiration, as well as slow fermentative growth at low temperature. In addition, coi1Δ cells harbour reduced steady-state levels of subunits of complexes III and IV as well as of the assembled complexes and supercomplexes. Interaction of Coi1 with respiratory chain subunits seems transient, as it appears to be a stoichiometric subunit neither of complex III nor of complex IV. Collectively, this work identifies a novel protein that plays a role in the assembly of the mitochondrial respiratory chain.

Correction for Khalimonchuk et al., "Formation of the Redox Cofactor Centers during Cox1 Maturation in Yeast Cytochrome Oxidase".

Correction for Khalimonchuk et al., "Formation of the Redox Cofactor Centers during Cox1 Maturation in Yeast Cytochrome Oxidase".

Identification of Oma1p Protease Sensitive Sites in Subunit 1 of Yeast Cytochrome Oxidase

Cytochrome c oxidase is the terminal electron acceptor in the electron transport chain in many aerobic organisms. This multi‐protein complex can be composed of a varying number of subunits depending on species. In the budding yeast, Saccharomyces cerevisiae, cytochrome c oxidase is made up of 11 subunits, 8 are derived from the nuclear genome and 3 are derived from the mitochondrial genome. To ensure these subunits come together at the correct time and place a family of assembly factors is required. In the case of the mitochondrial DNA encoded subunit 1 there is a linear pathway of biosynthesis where these assembly factors and other structural subunits associate at specific times to regulate specific maturation events. In the absence of some assembly factors subunit 1 is subject to degradation by the protease Oma1p. In yeast, this results in a deficiency in cytochrome c oxidase activity and the inability to grow using substrates requiring intact oxidative phosphorylation. We have performed a mutagenic screen of mitochondrial DNA to identify mutations in the subunit 1 gene that can suppress the oxidative phosphorylation deficient phenotype caused by the absence of the assembly factors, COA2 and SHY1. Through analysis of the COX1 gene we will identify putative regions required for the proteolytic processing of subunit 1 by the Oma1p protease. The information gathered from this research will guide us in identifying putative regions required for cytochrome c oxidase subunit 1 degradation and also provide more information regarding the proteolytic requirements of the Oma1p protease.

The Cox3p assembly module of yeast cytochrome oxidase

Yeast cytochrome oxidase (COX) was previously inferred to assemble from three modules, each containing one of the three mitochondrially encoded subunits and a different subset of the eight nuclear gene products that make up this respiratory complex. Pull-down assays of pulse-labeled mitochondria enabled us to characterize Cox3p subassemblies that behave as COX precursors and contain Cox4p, Cox7p, and Cox13p. Surprisingly, Cox4p is a constituent of two other complexes, one of which was previously proposed to be an intermediate of Cox1p biogenesis. This suggests that Cox4p, which contacts Cox1p and Cox3p in the holoenzyme, can be incorporated into COX by two alternative pathways. In addition to subunits of COX, some Cox3p intermediates contain Rcf1p, a protein associated with the supercomplex that stabilizes the interaction of COX with the bc1 (ubiquinol-cytochrome c reductase) complex. Finally, our results indicate that although assembly of the Cox1p module is not contingent on the presence of Cox3p, the converse is not true, as none of the Cox3p subassemblies were detected in a mutant blocked in translation of Cox1p. These studies support our proposal that Cox3p and Cox1p are separate assembly modules with unique compositions of ancillary factors and subunits derived from the nuclear genome.

Characterization of Assembly Intermediates Containing Subunit 1 of Yeast Cytochrome Oxidase

Mitochondrial-encoded Cox1p, one of the three core subunits of yeast cytochrome oxidase (COX), was previously shown to associate with regulatory proteins and nuclear-encoded subunits into five high molecular weight complexes that were proposed to constitute the pathway for biogenesis of the Cox1p assembly module. One of the intermediates (D5) was inferred, but not directly shown to exist. In the present study mitochondria of strains expressing C-terminal-tagged subunits of COX that had not been looked at previously were pulse-labeled and analyzed for the presence of newly translated Cox1p in the immunoprecipitates. These studies revealed that of the eight nuclear-encoded COX subunits, only Cox5ap, Cox6p, and Cox8p are present in the Cox1p module. Both Cox5ap and Cox8p share interfaces with Cox1p in the holoenzyme, whereas Cox6p interacts indirectly through Cox5ap. These results suggest that the subunit contacts in the holoenzyme are probably established during biogenesis of the Cox1p module. To confirm the existence of the largest Cox1p intermediates (D5), which was only inferred previously, radiolabeled Cox1p with a C-terminal tag was expressed in COX-deficient pet111 and pet494 mutants. Pulldown assays confirmed the presence of newly translated Cox1p in D5, which in wild type cannot be demonstrated directly because of its co-migration with COX in the native electrophoresis system used to separate the intermediates. Jointly, the results of these analyses substantiate our previous proposal that COX is assembled from separate assembly modules, each containing one of the mitochondrial-translated core subunits in association with a unique set of nuclear-encoded subunits.

Modular assembly of yeast cytochrome oxidase

Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high-molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.

Formation of the Redox Cofactor Centers during Cox1 Maturation in Yeast Cytochrome Oxidase

The biogenesis of cytochrome c oxidase initiates with synthesis and maturation of the mitochondrion-encoded Cox1 subunit prior to the addition of other subunits. Cox1 contains redox cofactors, including the low-spin heme a center and the heterobimetallic heme a(3):Cu(B) center. We sought to identify the step in the maturation of Cox1 in which the redox cofactor centers are assembled. Newly synthesized Cox1 is incorporated within one early assembly intermediate containing Mss51 in Saccharomyces cerevisiae. Subsequent Cox1 maturation involves the progression to downstream assembly intermediates involving Coa1 and Shy1. We show that the two heme a cofactor sites in Cox1 form downstream of Mss51- and Coa1-containing Cox1 intermediates. These Cox1 intermediates form normally in cells defective in heme a biosynthesis or in cox1 mutant strains with heme a axial His mutations. In contrast, the Shy1-containing Cox1 assembly intermediate is perturbed in the absence of heme a. Heme a(3) center formation in Cox1 appears to be chaperoned by Shy1. Cu(B) site formation occurs near or at the Shy1-containing Cox1 assembly intermediate also. The Cu(B) metallochaperone Cox11 transiently interacts with Shy1 by coimmunoprecipitation. The Shy1-containing Cox1 complex is markedly attenuated in cells lacking Cox11 but is partially restored with a nonfunctional Cox11 mutant. Thus, formation of the heterobimetallic Cu(B):heme a(3) site likely occurs in the Shy1-containing Cox1 complex.

Inhibition sites in F1-ATPase from bovine heart mitochondria

High-resolution crystallographic studies of a number of inhibited forms of bovine F1-ATPase have identified four independent types of inhibitory site: the catalytic site, the aurovertin B-binding site, the efrapeptin-binding site and the site to which the natural inhibitor protein IF1 binds. Hitherto, the binding sites for other inhibitors, such as polyphenolic phytochemicals, non-peptidyl lipophilic cations and amphiphilic peptides, have remained undefined. By employing multiple inhibition analysis, we have identified the binding sites for these compounds. Several of them bind to the known inhibitory sites. The amphiphilic peptides melittin and synthetic analogues of the mitochondrial import pre-sequence of yeast cytochrome oxidase subunit IV appear to mimic the natural inhibitor protein, and the polyphenolic phytochemical inhibitors resveratrol and piceatannol compete for the aurovertin B-binding site (or sites). The non-peptidyl lipophilic cation rhodamine 6G acts at a separate unidentified site, indicating that there are at least five inhibitory sites in the F1-ATPase. Each of the above inhibitors has significantly different activity against the bacterial Bacillus PS3 alpha3beta3gamma subcomplex compared with that observed with bovine F1-ATPase. IF1 does not inhibit the bacterial enzyme, even in the absence of the epsilon-subunit. An understanding of these inhibitors may enable rational development of therapeutic agents to act as novel antibiotics against bacterial ATP synthases or for the treatment of several disorders linked to the regulation of the ATP synthase, including ischaemia-reperfusion injury and some cancers.

Regulation of cytochrome c oxidase by adenylic nucleotides. Is oxidative phosphorylation feedback regulated by its end-products?

Cytochrome c oxidase, which catalyzes an irreversible step of the respiratory chain, is one of the rate-controlling steps of oxidative phosphorylation on isolated mitochondria. The rate of electron transfer through the complex is primarily controlled by the associated thermodynamic forces, i.e., the span in redox potential between oxygen and cytochrome c and the protonmotive force. However, the electron flux also depends on the various kinetic effectors, including adenylic nucleotides. Although the number of binding sites for ATP and ADP on cytochrome oxidase is still a matter of debate, experiments performed on the solubilized and reconstituted enzyme provide strong functional evidence that the mammalian cytochrome c oxidase binds adenylic nucleotides on both sides of the inner membrane. These effects include modification in cytochrome c affinity, allosteric inhibition and changes in proton pumping efficiency. Immunological studies have pointed out the role of subunit IV and that of an ATP-binding protein, subunit VIa, in these kinetic regulations. In yeast, the role of the nuclear-encoded subunits in assembly and regulation of the cytochrome c oxidase has been further substantiated by using gene-disruption analysis. Using a subunit VIa-null mutant, the consequences of the ATP regulation on oxidative phosphorylation have been further investigated on isolated mitochondria. Taken together, the data demonstrate that there are multiple regulating sites for ATP on the yeast cytochrome oxidase with respect to the location (matrix versus cytosolic side), kinetic effect (activation versus inhibition) and consequence on the flow-force relationships. The question is therefore raised as to the physiological meaning of such feedback regulation of the respiratory chain by ATP in the control and regulation of cellular energy metabolism.

CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro.

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.

Mitochondrial translation of Saccharomyces cerevisiae COX2 mRNA is controlled by the nucleotide sequence specifying the pre-Cox2p leader peptide.

The mitochondrial gene encoding yeast cytochrome oxidase subunit II (Cox2p) specifies a precursor protein with a 15-amino-acid leader peptide. Deletion of the entire leader peptide coding region is known to block Cox2p accumulation posttranscriptionally. Here, we examined in vivo the role of the pre-Cox2p leader peptide and the mRNA sequence that encodes it in the expression of a mitochondrial reporter gene, ARG8m, fused to the 91st codon of COX2. We found within the coding sequence antagonistic elements that control translation: the positive element includes sequences in the first 14 codons specifying the leader peptide, while the negative element appears to be within codons 15 to 91. Partial deletions, point mutations, and local frameshifts within the leader peptide coding region were placed in both the cox2::ARG8m reporter and in COX2 itself. Surprisingly, the mRNA sequence of the first six codons specifying the leader peptide plays an important role in positively controlling translation, while the amino acid sequence of the leader peptide itself is relatively unconstrained. Two mutations that partially block translation can be suppressed by nearby sequence substitutions that weaken a predicted stem structure and by overproduction of either the COX2 mRNA-specific translational activator Pet111p or the large-subunit mitochondrial ribosomal protein MrpL36p. We propose that regulatory elements embedded in the translated COX2 mRNA sequence could play a role, together with trans-acting factors, in coupling regulated synthesis of nascent pre-Cox2p to its insertion in the mitochondrial inner membrane.

Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase.

Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes. In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III. The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized. Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue. The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme. Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273-->Thr, Ile-280-->Thr and Gly-317-->Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (DeltaF102-F106). The subunit III mutation was highly deleterious and abolished enzyme assembly. The change Gly-317-->Ser had no effect on respiratory function. However, mutations Met-273-->Thr and Ile-280-->Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre.

Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase

Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes. In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III. The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized. Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue. The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme. Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273 → Thr, Ile-280 → Thr and Gly-317 → Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (∆F102–F106). The subunit III mutation was highly deleterious and abolished enzyme assembly. The change Gly-317 → Ser had no effect on respiratory function. However, mutations Met-273 → Thr and Ile-280 → Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre.

Identification of Cox20p, a Novel Protein Involved in the Maturation and Assembly of Cytochrome Oxidase Subunit 2

We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex. Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein. The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase. Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase. All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p. Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex. We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.

Isolation and characterization of Cox17p from porcine heart by determining its survival-promoting activity in NIH3T3 cells

We have found that the gel filtration fraction of porcine heart extract clearly promoted the survival of NIH3T3 fibroblast cells in the serum-free medium condition. A structural analysis showed that the active fraction contained a novel peptide, porcine Cox17p (p-Cox17p), which was recently reported by Chen et al. as dopuin (Z.W. Chen et al., Eur. J. Biochem. 249 (1997) 518–522). Porcine Cox17p/dopuin possesses high sequence homology to the product of human COX17 gene (h-Cox17p). Although Cox17p has been implied to be involved in copper recruitment to mitochondria and in the functional assembly of cytochrome oxidase in yeast, its role in mammalian cells is unknown. In this study, we chemically synthesized p-Cox17p to investigate its biological effects. Refolding experiments of synthesized linear p-Cox17p revealed the existence of mostly one pattern of three intrachain disulfide bridges similar to that of native p-Cox17p, because the main oxidized p-Cox17p was completely co-eluted with the natural product. The addition of heavy metal ions such as copper, zinc and cadmium significantly inhibited the formation of the oxidized form, suggesting that reduced p-Cox17p may interact directly with these metal ions. The reduced and oxidized forms of p-Cox17p were also confirmed to promote the survival of NIH3T3 cells in serum-free medium as observed with the natural product, indicating that Cox17p may be a bioactive peptide.

ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa.

The role of the nuclear-encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit VIa-null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP-regulating sites were characterized with respect to their location and their kinetic effect: (a) intra-mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of ATP on the wild-type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated by subunit VIa. However, at physiological ionic strength (i.e. approximately 200 mM), activation by ATP was absent but inhibition was not impaired; (b) in ethanol-respiring mitochondria, when the electron flux was modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild-type) or inhibition (null-mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of cytochrome oxidase on respiratory flux, decreased (wild-type) or increased (null-mutant) in the presence of ATP; (d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild-type but not for the mutant subunit. Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration. This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP.

Affinity Purification of Yeast Cytochrome Oxidase with Biotinylated Subunits 4, 5, or 6

Null mutants inCOX4, COX5a, orCOX6, which encode subunits 4, 5, and 6 of yeast cytochrome oxidase are blocked in assembly of the enzyme. The mutants are complemented by gene constructs expressing cytochrome oxidase subunits with a carboxyl terminal extension containing a biotinylation signal sequence. Spectra and enzyme activities of mitochondria from transformants expressing a biotinylated subunit indicate restoration of a functional cytochrome oxidase. Biotinylated cytochrome oxidase can be affinity-purified from mitochondrial extracts by fractionation on a monomeric avidin column. This method can be used to purify the enzyme from small amounts of starting material.