Coevolutionary Patterns in Cytochrome c Oxidase Subunit I Depend on Structural and Functional Context

The strength and pattern of coevolution between amino acid residues vary depending on their structural and functional environment. This context dependence, along with differences in analytical technique, is responsible for the different results among coevolutionary analyses of different proteins. It is thus important to perform detailed study of individual proteins to gain better insight into how context dependence can affect coevolutionary patterns even within individual proteins, and to unravel the details of context dependence with respect to structure and function. Here we extend our previous study by presenting further analysis of residue coevolution in cytochrome c oxidase subunit I sequences from 231 vertebrates using a statistically robust phylogeny-based maximum likelihood ratio method. As in previous studies, a strong overall coevolutionary signal was detected, and coevolution within structural regions was significantly related to the C_α distances between residues. While the strong selection for adjacent residues among predicted coevolving pairs in the surface region indicates that the statistical method is highly selective for biologically relevant interactions, the coevolutionary signal was strongest in the transmembrane region, although the distances between coevolving residues were greater. This indicates that coevolution may act to maintain more global structural and functional constraints in the transmembrane region. In the transmembrane region, sites that coevolved according to polarity and hydrophobicity rather than volume had a greater tendency to colocalize with just one of the predicted proton channels (channel H). Thus, the details of coevolution in cytochrome c oxidase subunit I depend greatly on domain structure and residue physicochemical characteristics, but proximity to function appears to play a critical role. We hypothesize that coevolution is indicative of a more important functional role for this channel. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Cytochrome cbo from the Obligate Methylotroph Methylobacillus flagellatus KT Is a Cytochrome c Oxidase

The cbo-type oxidase of Methylobacillus flagellatus KT was purified to homogeneity by preparative native gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cytochrome cbo with a pH optimum of 8.3. With TMPD as an electron donor for the cbo-type oxidase, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and only ascorbate. The kinetic constants determined at pH 7.0 were as follows: oxidation by the enzyme of reduced TMPD was characterized by K _M = 0.86 mM and V _max = 1.1 mol O₂/(min mg protein), and oxidation of reduced horse heart cytochrome c was characterized by K _M = 0.09 mM and V _max = 0.9 mol O₂/(min mg protein). Cyanide inhibited ascorbate/TMPD–oxidase activity (K _i = 4.5–5.0 M). The soluble cytochrome c _H (12 kDa), partially purified from M. flagellatus KT, was found to serve as a natural electron donor for the cbo-type oxidase.     

Evidence for a Conformational Change in Subunit III of Bovine Heart Mitochondrial Cytochrome c Oxidase1

The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl—biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.     

Human Cytochrome c Oxidase: Structure, Function, and Deficiency

As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear-encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either the mitochondrial or the nuclear genomes or both. A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA. Mutations of mtDNA appear, nonetheless, uncommon in early childhood. Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific nuclear genomic lesion has not yet been reported. Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice.     

The Profile of Cardiac Cytochrome c Oxidase (COX) Expression in an Accelerated Cardiac-Hypertrophy Model

Summary The contribution of the mitochondrial components, the main source of energy for the cardiac hypertrophic growth induced by pressure overload, is not well understood. In the present study, complete coarctation of abdominal aorta was used to induce the rapid development of cardiac hypertrophy in rats. One to two days after surgery, we observed significantly higher blood pressure and cardiac hypertrophy, which remained constantly high afterwards. We found an early increased level of cytochrome c oxidase (COX) mRNA determined by in-situ hybridization and dot blotting assays in the hypertrophied hearts, and a drop to the baseline 20 days after surgery. Similarly, mitochondrial COX protein level and enzyme activity increased and, however, dropped even lower than baseline 20 days following surgery. In addition, in natural hypertension-induced hypertrophic hearts in genetically hypertensive rats, the COX protein was significantly lower than in normotensive rats. Taken together, the lower efficiency of mitochondrial activity in the enlarged hearts of long-term complete coarcted rats or genetically hypertensive rats could be, at least partially, the cause of hypertensive cardiac disease. Additionally, the rapid complete coarctation-induced cardiac hypertrophy was accompanied by a disproportionate COX activity increase, which was suggested to maintain the cardiac energy-producing capacity in overloaded hearts.     

Interactions Between the Cytochrome Pathway and the Alternative Oxidase in Isolated Acanthamoeba castellanii Mitochondria

The steady-state activity of the two quinol-oxidizing pathways of Acanthamoeba castellanii mitochondria, the phosphorylating cytochrome pathway (i.e. the benzohydroxamate(BHAM)-resistant respiration in state 3) and the alternative oxidase (i.e. the KCN-resistant respiration), is shown to be fixed by ubiquinone (Q) pool redox state independently of the reducing substrate (succinate or exogenous reduced nicotinamide adenine dinucleotide (NADH)), indicating that the active Q pool is homogenous. For both pathways, activity increases with the Q reduction level (up to 80%). However, the cytochrome pathway respiration partially inhibited (about 50%) by myxothiazol decreases when the Q reduction level increases above 80%. The decrease can be explained by the Q cycle mechanism of complex III. It is also shown that BHAM has an influence on the relationship between the rate of ADP phosphorylation and the Q reduction level when alternative oxidase is active, and that KCN has an influence on the relationship between the alternative oxidase activity and the Q reduction level. These unexpected effects of BHAM and KCN observed at a given Q reduction level are likely due to functional connections between the two pathways activities or to protein–protein interaction.     

The Role of Electrostatic Interactions for Cytochrome c Oxidase Function

In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins. Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers. The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein. A cluster of 18 titratable groups around the heme a ₃–Cu_B binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site. Predicted changes in net protonation were in reasonable agreement with experimentally determined values. The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.     

A Theoretical Analysis of the Redox-Coupled Protonation of Heme a and CuA in the Cytochrome c Oxidase with CO-Inhibited Binuclear Center

One of the proposed mechanisms of functioning of cytochrome c oxidase (COX) postulates that hemea is the element pumping protons across the membrane. It is generally believed that, to support this mechanism, a substantial proton uptake/release should exist upon heme a reduction/oxidation. Two direct measurements of proton uptake/release in oxidation/reduction of heme a in CO-bound mixed-valence COX were recently reported. In this paper, we develop a general formalism for the interpretation of such experiments and discuss the results of these experiments. A control experiment is proposed to verify the conclusions made in previous studies.     

A Candidate Complex Approach to Study Functional Mitochondrial DNA Changes: Sequence Variation and Quaternary Structure Modeling of Drosophila simulans Cytochrome c Oxidase

A problem with studying evolutionary dynamics of mitochondrial (mt) DNA is that classical population genetic techniques cannot identify selected substitutions because of genetic hitchhiking. We circumvented this problem by employing a candidate complex approach to study sequence variation in cytochrome c oxidase (COX) genes within and among three distinct Drosophila simulans mtDNA haplogroups. First, we determined sequence variation in complete coding regions for all COX mtDNA and nuclear loci and their isoforms. Second, we constructed a quaternary structure model of D. simulans COX. Third, we predicted that six of nine amino acid changes in D. simulans mtDNA are likely to be functionally important. Of these seven, genetic crosses can experimentally determine the functional significance of three. Fourth, we identified two single amino acid changes and a deletion of two consecutive amino acids in nuclear encoded COX loci that are likely to influence cytochrome c oxidase activity. These data show that linking population genetics and quaternary structure modeling can lead to functional predictions of specific mtDNA amino acid mutations and validate the candidate complex approach. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Phylogeny of the Chipmunks Inferred from Mitochondrial Cytochrome b and Cytochrome Oxidase II Gene Sequences

There are currently 25 recognized species of the chipmunk genus Tamias. In this study we sequenced the complete mitochondrial cytochrome b (cyt b) gene of 23 Tamias species. We analyzed the cyt b sequence and then analyzed a combined data set of cyt b along with a previous data set of cytochrome oxidase subunit II (COII) sequence. Maximum-likelihood was used to further test the fit of models of evolution to the cyt b data. Other sciurid cyt b sequence was added to examine the evolution of Tamias in the context of other sciurids. Relationships among Tamias species are discussed, particularly the possibility of a current sorting event among taxa of the southwestern United States and the extreme divergences among the three subgenera (Neotamias, Eutamias, and Tamias).     

Kinetics of Electron Transfer within Cytochrome bc 1 and Between Cytochrome bc 1 and Cytochrome c

In this minireview an overview is presented of the kinetics of electron transfer within the cytochrome bc (1) complex, as well as from cytochrome bc (1) to cytochrome c. The cytochrome bc (1) complex (ubiquinone:cytochrome c oxidoreductase) is an integral membrane protein found in the mitochondrial respiratory chain as well as the electron transfer chains of many respiratory and photosynthetic bacteria. Experiments on both mitochondrial and bacterial cyatochrome bc (1) have provided detailed kinetic information supporting a Q-cycle mechanism for electron transfer within the complex. On the basis of X-ray crystallographic studies of cytochrome bc (1), it has been proposed that the Rieske iron-sulfur protein undergoes large conformational changes as it transports electrons from ubiquinol to cytochrome c (1). A new method was developed to study electron transfer within cytochrome bc (1) using a binuclear ruthenium complex to rapidly photooxidize cytochrome c (1). The rate constant for electron transfer from the iron-sulfur center to cytochrome c (1) was found to be 80,000 s(-1), and is controlled by the dynamics of conformational changes in the iron-sulfur protein. Moreover, a linkage between the conformation of the ubiquinol binding site and the conformational dynamics of the iron-sulfur protein has been discovered which could play a role in the bifurcated oxidation of ubiquinol. A ruthenium photoexcitation method has also been developed to measure electron transfer from cytochrome c (1) to cytochrome c. The kinetics of electron transfer are interpreted in light of a new X-ray crystal structure for the complex between cytochrome bc (1) and cytochrome c.     

Molecular Characterization of Taenia multiceps Isolates from Gansu Province,China by Sequencing of Mitochondrial Cytochrome C Oxidase Subunit 1

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.     

Structure of the Avian Mitochondrial Cytochrome bc 1 Complex

There are now four structures of vertebrate mitochondrial bc ₁ complexes available in theprotein databases and structures from yeast and bacterial sources are expected soon. Thisreview summarizes the new information with emphasis on the avian cytochrome bc ₁ complex(PDB entries 1BCC and 3BCC). The Rieske iron–sulfur protein is mobile and this has beenproposed to be important for catalysis. The binding sites for quinone have been located basedon structures containing inhibitors and, in the case of the quinone reduction site Qi, thequinone itself.     

Molecular Evolution at the Cytochrome Oxidase Subunit 2 Gene Among Divergent Populations of the Intertidal Copepod, Tigriopus californicus

The cytochrome c oxidase subunit 2 gene (COII) encodes a highly conserved protein that is directly responsible for the initial transfer of electrons from cytochrome c to cytochrome c oxidase (COX) crucial to the production of ATP during cellular respiration. Despite its integral role in electron transport, we have observed extensive intraspecific nucleotide and amino acid variation among 26 full-length COII sequences sampled from seven populations of the marine copepod, Tigriopus californicus. Although intrapopulation divergence was virtually nonexistent, interpopulation divergence at the COII locus was nearly 20% at the nucleotide level, including 38 nonsynonymous substitutions. Given the high degree of interaction between the cytochrome c oxidase subunit 2 protein (COX2) and the nuclear-encoded subunits of COX and cytochrome c (CYC), we hypothesized that some codons in the COII gene are likely to be under positive selection in order to compensate for amino acid substitutions in other subunits. Estimates of the ratio of nonsynonymous to synonymous substitution (ω), obtained using a series of maximum likelihood models of codon substitution, indicated that the majority of codons in T. californicus COII are under strong purifying selection (ω << 1), while approximately 4% of the sites in this gene appear to evolve under relaxed selective constraint (ω = 1). A branch-site maximum likelihood model identified three sites that may have experienced positive selection within the central California sequence clade in our COII phylogeny; these results are consistent with previous studies showing functional and fitness consequences among interpopulation hybrids between central and northern California populations. [Reviewing Editor: Dr. Willie Swanson]     

Reactivity of Nitric Oxide with Cytochrome c Oxidase: Interactions with the Binuclear Centre and Mechanism of Inhibition

Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the K _i being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized Cu_B leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from Cu_B to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.     

Molecular Phylogeny of the Chipmunk Genus Tamias Based on the Mitochondrial Cytochrome Oxidase Subunit II Gene

Complete sequences of the mitochondrial cytochrome oxidase subunit II gene were used to construct a phylogeny for 21 of the 25 currently recognized chipmunk species. Phylogenetic analyses indicate that T. striatus (subgenus Tamias, eastern United States) and T. sibiricus (subgenus Eutamias, Asia) are distantly related to the other species (subgenus Neotamias), which constitute a western North American radiation. We discuss and compare our molecular phylogeny to previous taxonomies and present a suggested classification of the species groups for the subgenus Neotamias.     

Cytochrome c Effect on Respiration of Heart Mitochondria: Influence of Various Factors

The effect of exogenous cytochrome c on respiration rate of the rat and human heart mitochondria was assessed in situ, using permeabilized fibers. It was (i) much more pronounced in State 2 and 4 than in State 3 with all the respiratory substrates (pyruvate+malate, succinate, palmitoyl-CoA+carnitine and octanoyl-L-carnitine), (ii) different with different substrates, (iii) much higher after ischemia in both metabolic states, particularly in the case of succinate oxidation compared to pyruvate+malate, (iv) the highest in State 4 with succinate as a substrate. Similar results were obtained with the isolated rat and rabbit heart mitochondria. The differences in the degree of stimulation of mitochondrial respiration by cytochrome c and, thus, sensitivity of cytochrome c test in evaluation of the intactness/injury of outer mitochondrial membrane are probably determined by the differences in the cytochrome c role in the control of mitochondrial respiration in the above-described conditions.     

Selective Cytochrome c Displacement by Phosphate and Ca2+ in Brain Mitochondria

In brain mitochondria, phosphate- and Ca²⁺-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca²⁺. Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2–[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC₁₂-HPC liposomes by either phosphate or Ca²⁺, but only Ca²⁺ produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Δψ_m) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca²⁺ caused inhibition of complex IV activity and impairment of Δψ_m. In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.     

Molecular Evolution of Cytochrome c Oxidase in High-Performance Fish (Teleostei: Scombroidei)

The 13 peptides encoded by vertebrate mitochondrial DNA (mtDNA) are essential subunits of oxidative phosphorylation (OXPHOS) enzymes. These genes normally experience purifying selection and also coevolve with nuclear-encoded subunits of OXPHOS complexes. However, the role of positive selection on mtDNA evolution is still unclear, as most examples of intergenomic coevolution appear to be the result of compensation by nuclear-encoded genes for mildly deleterious mtDNA mutations, and not simultaneous positive selection in both genomes. Organisms that have experienced strong selective pressures to increase aerobic capacity or adapt to changes in thermal environment may be better candidates in which to examine the impact of positively selected changes on mtDNA evolution. The tuna (suborder Scombroidei, family Scombridae) and billfish (suborder Scombroidei, families Xiphiidae and Istiophoridae) are highly aerobic fish with multiple specializations in muscle energetics, including a high mitochondrial content and regional endothermy. We examined the role of positively selected mtDNA substitutions in the production of these unique phenotypes. Focusing on a catalytic subunit of cytochrome c oxidase (COX II), we found that the rate ratio of nonsynonymous (d(N); amino acid changing)-to-synonymous (d(S); silent) substitutions was not increased in lineages leading to the tuna but was significantly increased in the lineage preceding the billfish. Furthermore, there are a number of individual positively selected sites that, when mapped onto the COX crystal structure, appear to interact with other COX subunits and may affect OXPHOS function and regulation in billfish.     

Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 microM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.