Rapid evolution of the human gene for cytochrome c oxidase subunit IV.

We have compared the DNA sequences of nine mammalian genes for cytochrome c oxidase subunit IV (COX4 genes)--four expressed genes (human, bovine, rat, and mouse) and five pseudogenes (human, chimpanzee, orangutan, squirrel monkey, and bovine)--and constructed the sequence of the ancestral mammalian COX4 gene. By analyzing these sequences to determine the pattern and rate of nucleotide substitution in each branch of the evolutionary tree, we deduced that the human gene has evolved rapidly since the origin of the primate pseudogene approximately 41 million years ago, and we discuss the suggestion that this results from coevolution of nuclear and mitochondrial genes for cytochrome c oxidase.     

Oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase.

Attempts to rationalize the kinetics of cytochrome c oxidation catalyzed by solubilized mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) have been based on assumptions of productive complex formation (Michaelis-Menten approach). However, the range of substrate concentrations used has not, in general, been sufficient to establish a general rate equation. Data adequate to derive such a rate expression are presented, as well as a method for estimation of constants which appear in the rate law deduced and reported herein. It is shown that either of two types of mechanisms, one assuming productive complex formation, as opposed to the other postulating dead-end complex formation, accurately predict the rate equation as deduced from experiment.     

Coupling in cytochrome c oxidase

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K(+) when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome c and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid.     

The mechanism of proton pumping by cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water that is accompanied by pumping of four protons across the mitochondrial or bacterial membrane. Triggered by the results of recent x-ray crystallographic analyses, published data concerning the coupling of individual electron transfer steps to proton pumping are reanalyzed: Conversion of the conventional oxoferryl intermediate F to the fully oxidized form O is connected to pumping of only one proton. Most likely one proton is already pumped during the double reduction of O, and only three protons during conversion of the “peroxy” forms P to O via the oxoferryl form F. Based on the available structural, spectroscopic, and mutagenesis data, a detailed mechanistic model, carefully considering electrostatic interactions, is presented. In this model, each of the four reductions of heme a during the catalytic cycle is coupled to the uptake of one proton via the D-pathway. These protons, but never more than two, are temporarily stored in the regions of the heme a and a₃ propionates and are driven to the outside (“pumped”) by electrostatic repulsion from protons entering the active site during turnover. The first proton is pumped by uptake of one proton via the K-pathway during reduction, the second and third proton during the P → F transition when the D-pathway and the active site become directly connected, and the fourth one upon conversion of F to O. Atomic structures are assigned to each intermediate including F′ with an alternative route to O.     

Impaired redox status and cytochrome c oxidase deficiency in patients with polymyalgia rheumatica

OBJECTIVE: To evaluate redox status and muscular mitochondrial abnormalities in patients with polymyalgia rheumatica (PMR). METHODS: Prospective evaluation of deltoid muscle biopsy in 15 patients with PMR. Fifteen subjects matched for age and sex, with histologically normal muscle and without clinical evidence of myopathy, were used as controls. Cryostat sections of muscle were processed for conventional dyes, cytochrome c oxidase (COX), usual histochemical reactions, and Sudan black. A total of 300-800 fibres was examined in each case. Blood lactate, pyruvate, and lactate/pyruvate ratio were determined in all patients. RESULTS: Ragged red fibres were found in eight patients with PMR and accounted for 0-0.5% of fibres. Focal COX deficiency was found in 14 (93%) of 15 patients and in nine (60%) of 15 controls. COX deficient fibres were more common in patients with PMR (range 0-2.5%; mean 0.9%) than in controls (range 0-1.2%; mean 0.3%) (paired t test, p=0.001). Seven (47%) of 15 patients had high blood lactate levels (1.50-2.60 mmol/l) or high blood lactate/pyruvate ratios (22-25). CONCLUSIONS: PMR is associated with mitochondrial abnormalities not solely related to the aging process.     

Functional coadaptation between cytochrome c and cytochrome c oxidase within allopatric populations of a marine copepod

Geographically isolated populations may accumulate alleles that function well on their own genetic backgrounds but poorly on the genetic backgrounds of other populations. Consequently, interpopulation hybridization may produce offspring of low fitness as a result of incompatibilities arising in allopatry. Genes participating in these epistatic incompatibility systems remain largely unknown. In fact, despite the widely recognized importance of epistatic interactions among gene products, few data directly address the functional consequences of such interactions among natural genetic variants. In the marine copepod, Tigriopus californicus, we found that the cytochrome c variants isolated from two different populations each had significantly higher activity with the cytochrome c oxidase derived from their respective source population. Three amino acid substitutions in the cytochrome c protein appear to be sufficient to confer population specificity. These results suggest that electron transport system (ETS) proteins form coadapted sets of alleles within populations and that disruption of the coadapted ETS gene complex leads to functional incompatibilities that may lower hybrid fitness.     

Conformational switching at cytochrome a during steady-state turnover of cytochrome c oxidase.

As an electron transfer-driven proton pump, cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) must alternate between two conformations in each valence state of the redox element associated with ion translocation. Using second derivative absorption spectroscopy, the conformation of the cytochrome a cofactor has been investigated during steady-state turnover of this enzyme. Resting cytochrome c oxidase displays a transition for ferric cytochrome a at 430 nm. During aerobic steady-state turnover, this band is replaced by a ferrous cytochrome a transition at 450 nm. When anaerobicity is achieved, the transition occurs at 444 nm. The 450-nm-absorbing species is thus the dominant form during turnover, suggesting that conformational transitions of cytochrome a direct electron transfer during catalysis and may direct as well proton translocation in the last step of the respiratory electron transfer chain.     

Interheme electron tunneling in cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain that catalyzes respiratory reduction of dioxygen (O(2)) to water in all eukaryotes and many aerobic bacteria. CcO, and its homologs among the heme-copper oxidases, has an active site composed of an oxygen-binding heme and a copper center in the vicinity, plus another heme group that donates electrons to this site. In most oxidoreduction enzymes, electron transfer (eT) takes place by quantum-mechanical electron tunneling. Here we show by independent molecular dynamics and quantum-chemical methods that the heme-heme eT in CcO differs from the majority of cases in having an exceptionally low reorganization energy. We show that the rate of interheme eT in CcO may nevertheless be predicted by the Moser-Dutton equation if reinterpreted as the average of the eT rates between all individual atoms of the donor and acceptor weighed by the respective packing densities between them. We argue that this modification may be necessary at short donor/acceptor distances comparable to the donor/acceptor radii.     

Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase

Elucidating the structures of intermediates in the reduction of O₂ to water by cytochrome c oxidase is crucial to understanding both oxygen activation and proton pumping by the enzyme. In the work here, the reaction of O₂ with the mixed-valence enzyme, in which only heme a₃ and Cu_B in the binuclear center are reduced, has been followed by time-resolved resonance Raman spectroscopy. The results show that O=O bond cleavage occurs within the first 200 μs after reaction initiation; the presence of a uniquely stable Fe—O—O(H) peroxy species is not detected. The product of this rapid reaction is a heme a₃ oxoferryl (Fe^IV=O) species, which requires that an electron donor in addition to heme a₃ and Cu_B must be involved. The available evidence suggests that the additional donor is an amino acid side chain. Recent crystallographic data [Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Libeu, C. P., Mizushima, T., et al. Science, in press; Ostermeier, C., Harrenga, A., Ermler, U. & Michel, H. (1997) Proc. Natl. Acad. Sci. USA 94, 10547–10553] show that one of the Cu_B ligands, His240, is cross-linked to Tyr244 and that this cross-linked tyrosyl is ideally positioned to participate in dioxygen activation. We propose a mechanism for O—O bond cleavage that proceeds by concerted hydrogen atom transfer from the cross-linked His—Tyr species to produce the product oxoferryl species, Cu_B²⁺—OH⁻, and the tyrosyl radical. This mechanism provides molecular structures for two key intermediates that drive the proton pump in oxidase; moreover, it has clear analogies to the proposed O—O bond forming chemistry that occurs during O₂ evolution in photosynthesis.     

Biosynthesis of polypeptides of cytochrome c oxidase by isolated mitochondria.

Yeast mitochondria, incubated with radioactive amino acids in a "protein-synthesizing mixture" containing an oxidizable substrate and an ATP regenerating system, have been shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to incorporate label into polypeptides equivalent in molecular weight and relative amount ot those made in vivo in the presence of cycloheximide. The ability of these isolated mitochondria to synthesize "native" polypeptides was assessed by examining the incorporation of label into subunits of cytochrome c oxidase (EC 1.9.3.1). An analysis of immunoprecipitates formed by incubating cholate extracts of labeled mitochondria with an antiserum against holocytochrome c oxidase revealed that label was incorporated into three polypeptides of sizes equivalent to those of cytochrome c oxidase subunits I, II, and III, shown from earlier studies in vivo to be translated on mitochondrial ribosomes. Further evidence that these polypeptides made in vitro are "native" and identical to subunits I, II, and III was provided by the observation that labeled polypeptides equivalent in size to subunits I-III- ARE ALSO IMMUNO-PRECIPITATED BY ANTISERUM AGAINST SUBUNITS V plus VII, an antiserum that can precipitate subunits I, II, and III only when they are complexed to the cytoplasmically synthesized subunits, V and VII, of the enzyme. These results suggest that isolated mitochondria are capable of synthesizing three subunits of cytochrome c oxidase and assembling them into a holoenzyme.     

Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2.     

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is investigated by (1)H-(15)N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt c induced by addition of unlabeled CcO indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt c dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt c to provide well controlled ET. Upon oxidation of Cyt c, the specific line broadening of N-H signals disappeared and high field (1)H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with CcO are reduced by steric constraint in oxidized Cyt c, while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt c with CcO. These results suggest that the overall affinity of oxidized Cyt c for CcO is significantly, but not very much weaker than that of reduced Cyt c. Thus, electron transfer is gated by dissociation of oxidized Cyt c from CcO, the rate of which is controlled by the affinity of oxidized Cyt c to CcO for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon CcO binding to oxidized Cyt c, shown by (1)H- and (1)H, (15)N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme c environment.     

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm(-1) range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(-), and 3,607(+) cm(-1). A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D(2)O and H(2)(18)O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.     

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Well ordered reproducible crystals of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides yield a previously unreported structure at 2.0 A resolution that contains the two catalytic subunits and a number of alkyl chains of lipids and detergents. Comparison with crystal structures of other bacterial and mammalian CcOs reveals that the positions occupied by native membrane lipids and detergent substitutes are highly conserved, along with amino acid residues in their vicinity, suggesting a more prevalent and specific role of lipid in membrane protein structure than often envisioned. Well defined detergent head groups (maltose) are found associated with aromatic residues in a manner similar to phospholipid head groups, likely contributing to the success of alkyl glycoside detergents in supporting membrane protein activity and crystallizability. Other significant features of this structure include the following: finding of a previously unreported crystal contact mediated by cadmium and an engineered histidine tag; documentation of the unique His-Tyr covalent linkage close to the active site; remarkable conservation of a chain of waters in one proton pathway (D-path); and discovery of an inhibitory cadmium-binding site at the entrance to another proton path (K-path). These observations provide important insight into CcO structure and mechanism, as well as the significance of bound lipid in membrane proteins.     

The proton donor for O-O bond scission by cytochrome c oxidase

Cytochrome c oxidase is the main catalyst of oxygen consumption in mitochondria and many aerobic bacteria. The key step in oxygen reduction is scission of the O-O bond and formation of an intermediate P(R) of the binuclear active site composed of heme a(3) and Cu(B). The donor of the proton required for this reaction has been suggested to be a unique tyrosine residue (Tyr-280) covalently cross-linked to one of the histidine ligands of Cu(B). To test this idea we used the Glu-278-Gln mutant enzyme from Paracoccus denitrificans, in which the reaction with oxygen stops at the P(R) intermediate. Three different time-resolved techniques were used. Optical spectroscopy showed fast (approximately 60 micros) appearance of the P(R) species along with full oxidation of heme a, and FTIR spectroscopy revealed a band at 1,308 cm(-1), which is characteristic for the deprotonated form of the cross-linked Tyr-280. The development of electric potential during formation of the P(R) species suggests transfer of a proton over a distance of approximately 4 A perpendicular to the membrane plane, which is close to the distance between the oxygen atom of the hydroxyl group of Tyr-280 and the bound oxygen. These results strongly support the hypothesis that the cross-linked tyrosine is the proton donor for O-O bond cleavage by cytochrome c oxidase and strengthens the view that this tyrosine also provides the fourth electron in O(2) reduction in conditions where heme a is oxidized.     

Interaction of nitric oxide with a functional model of cytochrome c oxidase

Cytochrome c oxidase (CcO) is a multimetallic enzyme that carries out the reduction of O2 to H2O and is essential to respiration, providing the energy that powers all aerobic organisms by generating heat and forming ATP. The oxygen-binding heme a(3) should be subject to fatal inhibition by chemicals that could compete with O2 binding. Near the CcO active site is another enzyme, NO synthase, which produces the gaseous hormone NO. NO can strongly bind to heme a(3), thus inhibiting respiration. However, this disaster does not occur. Using functional models for the CcO active site, we show how NO inhibition is avoided; in fact, it is found that NO can protect the respiratory enzyme from other inhibitors such as cyanide, a classic poison.     

Copper electron-nuclear double resonance of cytochrome c oxidase.

Electron-nuclear double resonance of copper was observed while monitoring the "intrinsic copper" electron paramagnetic resonance signal of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) near g = 2. This unambiguously establishes the presence of the metal (Cua) in the redox center responsible for this signal. The hyperfine couplings to copper are largely istropic and the maximum value is about half that seen in type I blue copper proteins. The magnetic properties of this oxidized copper center are not consistent with those of a thiyl radical (R-S) coordinated to Cu(I), and thus favor the identification of this redox center as a Cu(II) ion in a unique environment.     

Initiation of the proton pump of cytochrome c oxidase

Cytochrome c oxidase is the terminal enzyme of the respiratory chain that is responsible for biological energy conversion in mitochondria and aerobic bacteria. The membrane-bound enzyme converts free energy from oxygen reduction to an electrochemical proton gradient by functioning as a redox-coupled proton pump. Although the 3D structure and functional studies have revealed proton conducting pathways in the enzyme interior, the location of proton donor and acceptor groups are not fully identified. We show here by time-resolved optical and FTIR spectroscopy combined with time-resolved electrometry that some mutant enzymes incapable of proton pumping nevertheless initiate catalysis by proton transfer to a proton-loading site. A conserved tyrosine in the so-called D-channel is identified as a potential proton donor that determines the efficiency of this reaction.