Cytochrome c oxidase, Cu,Zn-superoxide dismutase, and ceruloplasmin activities in copper-deficient bovines

The activity of several cuproenzymes in relation to the immune system was examined in serum and blood cells from bovines with molybdenum-induced copper deficiency. Five female cattle were given molybdenum (30 ppm) and sulfate (225 ppm) to induce experimental secondary copper deficiency. Ceruloplasmin activity was determined in serum. The Cu,Zn-superoxide dismutase and cytochrome c oxidase activities were measured in peripheral blood lymphocytes, neutrophils, and monocyte-derived macrophages. Copper deficiency was confirmed from decreased serum copper levels and the animals with values less than 5.6 μmol/L were considered deficient. The content of intracellular copper decreased between 40% and 70% in deficient cells compared with the controls. In copper-deficient animals, the serum ceruloplasmin activity decreased to half of the control value. Both of them, the Cu,Zn-superoxide dismutase and the cytochrome c oxidase activities, undergo a significant reduction in leukocytes, showing differences among diverse cell populations. We concluded that the copper deficiency alters the activity of several enzymes, which mediate antioxidant defenses and ATP formation. These effects may impair the cell immune functionality, affecting the bactericidal capacity and making the animals more susceptible to infection.     

Copper-induced trafficking of the Cu-ATPases: A key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (ccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement ccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.     

Overlap of copper and iron uptake systems in mitochondria in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix.     

Morphological development and cytochrome c oxidase activity in Streptomyces lividans are dependent on the action of a copper bound Sco protein

Copper has an important role in the life cycle of many streptomycetes, stimulating the developmental switch between vegetative mycelium and aerial hyphae concomitant with the production of antibiotics. In streptomycetes, a gene encoding for a putative Sco-like protein has been identified and is part of an operon that contains two other genes predicted to handle cellular copper. We report on the Sco-like protein from Streptomyces lividans (Sco^Sl) and present a series of experiments that firmly establish a role for Sco^Sl as a copper metallochaperone as opposed to a role as a thiol-disulphide reductase that has been assigned to other bacterial Sco proteins. Under low copper concentrations, a Δsco mutant in S. lividans displays two phenotypes; the development switch between vegetative mycelium and aerial hyphae stalls and cytochrome c oxidase (CcO) activity is significantly decreased. At elevated copper levels, the development and CcO activity in the Δsco mutant are restored to wild-type levels and are thus independent of Sco^Sl. A CcO knockout reveals that morphological development is independent of CcO activity leading us to suggest that Sco^Sl has at least two targets in S. lividans. We establish that one Sco^Sl target is the dinuclear Cu_A domain of CcO and it is the cupric form of Sco^Sl that is functionally active. The mechanism of cupric ion capture by Sco^Sl has been investigated, and an important role for a conserved His residue is identified.     

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.     

Genomic analysis reveals widespread occurrence of new classes of copper nitrite reductases

Recently, the structure of a Cu-containing nitrite reductase (NiR) from Hyphomicrobium denitrificans (HdNiR) has been reported, establishing the existence of a new family of Cu-NiR where an additional type 1 Cu (T1Cu) containing cupredoxin domain is located at the N-terminus (Nojiri et al. in Proc. Natl. Acad. Sci. USA 104:4315-4320, 2007). HdNiR retains the well-characterised coupled T1Cu-type 2 Cu (T2Cu) core, where the T2Cu catalytic site is also built utilising ligands from neighbouring monomers. We have undertaken a genome analysis and found the wide occurrence of these NiRs, with members clustering in two groups, one showing an amino acid sequence similarity of around 80% with HdNiR, and a second group, including the NiR from the extremophile Acidothermus cellulolyticus, clustering around 50% similarity to HdNiR. This is reminiscent of the difference observed between the blue (Alcaligenes xylosoxidans) and green (Achromobacter cycloclastes and Alcaligenes faecalis) NiRs which have been extensively studied and may indicate that these also form two distinct subclasses of the new family. Genome analysis also showed the presence of Cu-NiRs with a C-terminal extension of 160-190 residues containing a class I cytochrome c domain with a characteristic beta-sheet extension. Currently no structural information exists for any member of this family. Genome analysis suggests the widespread occurrence of these novel NiRs with representatives in the alpha, beta and gamma subclasses of the Proteobacteria and in two species of the fungus Aspergillus. We selected the enzyme from Ralstonia pickettii for comparative modelling and produced a plausible structure highlighting an electron transfer mode in which the cytochrome c haem at the C-terminus can come within 16-A reach of the T1Cu centre of the T1Cu-T2Cu core.     

Identification of deficient mitochondrial signaling in apoptosis resistant leukemia cells by flow cytometric analysis of intracellular cytochrome c, caspase-3 and apoptosis

Deficient activation of apoptosis signaling pathways may be responsible for treatment failure of malignant diseases. In primary leukemia samples the detection of deficient mitochondrial apoptosis signaling would enable identification of chemo-resistant cells. To investigate the key events of apoptosis at the mitochondrial level, we developed a flow cytometric method for simultaneous detection of mitochondrial cytochrome c release and caspase-3 processing using conformation sensitive monoclonal antibodies. This method proved to identify deficient mitochondrial apoptosis signaling in leukemia cells overexpressing Bcl-2 by a pattern of apoptosis resistance, deficient cytochrome c reduction and partial processing of caspase-3. In primary leukemia cells, reduction of cytochrome c and caspase-3 activation was induced by treatment with anticancer drugs in vitro. In leukemia cells of a patient with resistant disease, a pattern of deficient apoptosis signaling as in Bcl-2 transfected cells was observed, suggesting that deficient mitochondrial signaling contributed to the clinical phenotype of drug resistance in this patient. Flow cytometric analysis of mitochondrial apoptosis signaling may provide a useful tool for the prediction of drug resistance and treatment failure in primary leukemia.     

Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.     

A comparison of catalytic site intermediates of cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase and peroxidases

Compounds I and II of peroxidases such as horseradish peroxidase and cytochrome c peroxidase are relatively well understood catalytic intermediates in terms of their structures and redox states of iron, heme, and associated radical species. The intermediates involved in the oxygen reduction chemistry of the cytochrome c oxidase superfamily are more complicated because of the need for four reducing equivalents and because of the linkage of the oxygen chemistry with vectorial proton translocations. Nevertheless, two of these intermediates, the peroxy and ferryl forms, have characteristics that can in many ways be considered to be counterparts of peroxidase compounds I and II. We explore the primary factors that minimize the generation of unwanted reactive oxygen species products and ensure that the principal enzymological function becomes either that of a peroxidase or an oxidase. These comparisons can provide insights into the nature of biological oxygen reduction chemistry and guidance for the engineering of biomimetic synthetic materials. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 10, pp. 1289–1299.     

Diethyldithiocarbamate inhibits in vivo Cu,Zn-superoxide dismutase and perturbs free radical processes in the yeast Saccharomyces cerevisiae cells

Copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in some model experiments in vitro demonstrated antioxidant as well as pro-oxidant properties. In the present study, yeast Saccharomyces cerevisiae lacking Mn-SOD were studied using Cu,Zn-SOD inhibitor N-N'-diethyldithiocarbamate (DDC) as a model system to study the physiological role of the yeast Cu,Zn-SOD. Yeast treatment by DDC caused dose-dependent inhibition of SOD in vivo, with 75% inhibition at 10mM DDC. The inhibition of SOD by DDC resulted in modification of carbonylprotein levels, indicated by a bell-shaped curve. The activity of glutathione reductase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase (enzymes associated with antioxidant) increased, demonstrating a compensatory effect in response to SOD inhibition by different concentrations of DDC. A strong positive correlation (R2=0.97) was found between SOD and catalase activities that may be explained by the protective role of SOD for catalase. All observed effects were absent in the isogenic SOD-deficient strain that excluded direct DDC influence. The results are discussed from the point of view that in vivo Cu,Zn-SOD of S. cerevisiae can demonstrate both anti- and pro-oxidant properties.     

Copper-binding proteins and copper tolerance in Pisam sativum L.

The effect of high nutrient levels of copper on the low-molecular-weight copper-proteins of leaves from plants of two cultivars of Pisum sativum L., with different sensitivity to copper, was investigated. Gel-filtration chromatography of leaf extracts from Cu-tolerant and Cu-sensitive plants grown with 1 M Cu(II), showed the presence of only two copper peaks (I and II), but growth of plants with 240 M Cu(II) induced two additional copper fractions (III and IV). Fractions II and III were purified by solvent extraction, gel-filtration and ion-exchange chromatography, and their molecular weights, subunit sizes, absorption spectra, metalprotein stoichiometry and amino-acid contents were determined. Fraction II was a polypeptide of M_r 15000 composed of a single chain. The purification of fraction III produced a copper-containing fraction (III-1) of M_r 3700, and a copper-protein (III-2) with an M_r, by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, of 66000. The metal contents of fractions III-1 and III-2 were higher in Cu-tolerant than in Cu-sensitive plants. On the basis of amino-acid analyses, fraction III-1 appeared to be complexes of Cu(II)-poly-isoleucine and Cu(II)-poly-leucine. The results rule out the existence, in pea leaves, of any protein similar to either animal metallothioneins or to any of the low-molecularweight metal-binding proteins or peptides described in other plants and reported to be involved in metal tolerance. In the mechanism of copper tolerance at the leaf level, fractions III-1 (M_r 3700), III-2 (M_r 66000), and IV (M_r 2000) appear to have a role, fraction IV being specifically induced in the tolerant cultivar by Cu(II). Fractions III-1 and III-2 could participate in a different mechanism, adaptive in character, involving an enhanced capacity to bind copper in Cu-tolerant plants.Abbreviations DEAE diethylaminoethyl - M_r relative molecular mass - SDS sodium dodecyl sulfate - PAGE polyacrylamide-gel electrophoresis J.M. Palma was the recipient of a research fellowship from the Caja General de Ahorros y Monte de Piedad de Granada and CSIC. We are grateful to Dr. J. Moreno-Carretero, R + D Department, UNIASA, Granada, for conducting the amino-acid analyses. This work was supported by grant 603/275 from CAICYT-CSIC (Spain).     

Role of cooperative H(+)/e(-) linkage (redox bohr effect) at heme a/Cu(A) and heme a(3)/Cu(B) in the proton pump of cytochrome c oxidase

It is a pleasure to contribute to the special issue published in honor of Vladimir Skulachev, a distinguished scientist who greatly contributes to maintain a high standard of biochemical research in Russia. A more particular reason can be found in his work (Artzabanov, V. Y., Konstantinov, A. A., and Skulachev, V. P. (1978) FEBS Lett., 87, 180–185), where observations anticipating some ideas presented in my article were reported. Cytochrome c oxidase exhibits protonmotive, redox linked allosteric cooperativity. Experimental observations on soluble bovine cytochrome c oxidase are presented showing that oxido-reduction of heme a/Cu_A and heme a ₃/Cu_B is linked to deprotonation/protonation of two clusters of protolytic groups, A₁ and A₂, respectively. This cooperative linkage (redox Bohr effect) results in the translocation of 1 H⁺/oxidase molecule upon oxido-reduction of heme a/Cu_A and heme a ₃/Cu_B, respectively. Results on liposome-reconstituted oxidase show that upon oxidation of heme a/Cu_A and heme a ₃/Cu_B protons from A₁ and A₂ are released in the outer aqueous phase. A₁ but not A₂ appears to take up protons from the inner aqueous space upon reduction of the respective redox center. A cooperative model is presented in which the A₁ and A₂ clusters, operating in close sequence, constitute together the gate of the proton pump in cytochrome c oxidase.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 220–230.Original Russian Text Copyright © 2005 by Papa.This revised version was published online in April 2005 with corrections to the post codes.     

Identification of a copper transporter family in Arabidopsis thaliana

Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes. In this work, a five-member family (COPT1–5) of putative Arabidopsis copper transporters is described. We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant. The specific expression pattern of the Arabidopsis COPT1–5 mRNA in different tissues was analyzed by RT-PCR. Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed. Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S. cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells.     

Copper nutrition of Eucalyptus maculata Hook. seedlings: Requirements for growth,distribution of copper and the diagnosis of copper deficiency

A glasshouse experiment was conducted to define the response of Eucalyptus maculata seedlings to the addition of nine rates of copper (Cu) to a Cu-deficient sand. Plants were harvested 128 days from sowing. Symptoms of Cu deficiency included marginal necrosis in young fully expanded leaves (YFEL), deformed leaf margins, death of lateral shoots, bleeding at nodes on the main stem and reduced lignification of xylem fibres and vessels. Plant height and the number of nodes on the main stem were unaffected. In plants supplied with 0 Cu, whole top and root fresh weights were depressed by 27% and 32% respectively. The external Cu requirement for maximum growth of E. maculata seedlings was similar to that for wheat grown in the same soil. In Cu-adequate plants, leaf Cu concentrations decreased with distance from the shoot apex. Cu levels in stems varied little with position and were similar to the YFEL. Cu concentrations in leaves and stems were depressed in Cu-deficient plants to <1.0 g g^–1 dry weight (d.w.) (roots: 1.5 g g^–1 d.w.). The external Cu supply did not greatly alter the distribution of Cu within the plant. Young leaves at the shoot tip are recommended for diagnosis of Cu deficiency: critical values for shoot d.w. were about 1.5 g Cu g^–1 d.w. Lignification of wood was suppressed where Cu concentrations fell below 1.5 g g^–1 d.w.: the Bussler test for lignification would thus be a valuable indicator of Cu deficiency.     

Zinc ions as cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase inhibitors: two sites of action

Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix. This interaction is fully reversed by EDTA and results in a partial inhibition of the enzyme activity (50–90%,depending on preparation) with an effective K _i of 10 µM. The rapid effect of zinc is observed with the solubilized enzyme, it vanishes upon incorporation of cytochrome oxidase in liposomes,and it re-appears when proteoliposomes are supplied with alamethicin that makes the membrane permeable to low molecular weight substances. Zinc presumably blocks the entrance of the D-protonic channel opening into the inner aqueous phase. Second, zinc interacts slowly (tens of minutes, hours) with a site of cytochrome oxidase accessible from the outer aqueous phase bringing about complete inhibition of the enzymatic activity. The slow phase is characterized by high affinity of the inhibitor for the enzyme:full inhibition can be achieved upon incubation of the solubilized oxidase for 24 h with zinc concentration as low as 2 µM. The rate of zinc inhibitory action in the slow phase is proportional to Zn²⁺ concentration. The slow interaction of zinc with the outer surface of liposome-reconstituted cytochrome oxidase is observed only with the enzyme turning over or in the presence of weak reductants, whereas incubation of zinc with the fully oxidized proteoliposomes does not induce the inhibition. It is shown that zinc ions added to cytochrome oxidase proteoliposomes from the outside inhibit specifically the slow electrogenic phase of proton transfer, coupled to a transition of cytochrome oxidase from the oxo-ferryl to the oxidized state (the F O step corresponding to transfer of the 4th electron in the catalytic cycle).Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 160–170.Original Russian Text Copyright © 2005 by Kuznetsova, Azarkina, Vygodina, Siletsky, Konstantinov.This revised version was published online in April 2005 with corrections to the post codes.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

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.     

Methionine-oxidized horse heart cytochromes c. I. Reaction with Chloramine-T, products, and their oxidoreduction properties

Spectroscopically, the modification of horse heart ferricytochrome c with N-chloro-4-toluolsul-fonamide (Chloramine-T, CT) occurs through a two-step process, the disruption of the methionine-80 sulfur-iron linkage and a reagent-independent change, an intramolecular rearrangement. Chromatographic purification of the preparation at a 2.5:1 reagent-to-protein ratio, pH 8.0–8.5, yields two major products, the F^II and F^III CT-cytochromes c. Both products contain modification of only the methionines, 80 and 65, to sulfoxides; both are monomeric, reduced by ascorbate, and the ferrous forms are oxidized by molecular oxygen and bind carbon monoxide. The redox potentials of F^II and F^III are 135 and 175±15 mV. The F^III is indistinguishable from the native protein in its binding and the electron donor property toward mammalian cytochrome c oxidase. It also binds nearly as effectively as the native protein to yeast cytochrome c peroxidase, but is a less efficient donor. It is, however, a poor electron acceptor from both mammalian cytochrome c reductase and chicken liver sulfite oxidase. F^II lacks cytochrome c oxidase activity and is also a poorer substrate for the other three enzymes. Both the derivatives are consistently better electron donors than acceptors. It is concluded that the binding of cytochrome c to cytochrome c oxidase and to cytochrome c peroxidase does not require the integrity of the methionine-80 sulfur linkage and that the complexation process has a finite degree of freedom with regard to the state of the heme crevice opening. The alterations of the oxidoreduction function have been analyzed in light of both prevailing models of cytochrome c function, the two-site model (one site for oxidizing and the other for reducing enzymes) and the single-site model (the same site for the oxidizing and reducing enzymes). These observations can be accommodated by either model, given the latitude that the binding domains for the oxidizing and the reducing enzymes have finite overlapping and nonoverlapping regions.To whom all correspondence related to the functional studies with cytochrome c peroxidase and sulfite oxidase is to be directed.     

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.