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Biomedical subjects

N Capitanio

Publications and source records attributed to N Capitanio.

At least 19 recordsLinked to original sources
PubMed2006

Mitochondrial dysfunction in hepatitis C virus infection.

The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood though HCV induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory diseases. This mini-review will focus on recent findings revealing an unexpected role of mitochondria in providing a central role in the innate immunity and in addition will illustrate the application of stably transfected human-derived cell lines, inducibly expressing the entire HCV open reading frame for in vitro studies on mitochondria. Results obtained by a comparative analysis of the respiratory chain complexes activities along with mitochondrial morpho-functional confocal microscopy imaging show a detrimental effect of HCV proteins on the cell oxidative metabolism with specific inhibition of complex I activity, decrease of mtDeltaPsi, increased production of reactive oxygen species. A possible de-regulation of calcium recycling between the endoplasmic reticulum and the mitochondrial network is discussed to provide new insights in the pathogenesis of hepatitis C.

Gene Expression Regulation, Viral↗
PubMed2000

The proton/electron coupling ratio at heme a and Cu(A) in bovine heart cytochrome c oxidase.

Measurements of the H(+)/heme a, Cu(A) ratios for proton-electron coupling at these centers (redox Bohr effect) in CO-inhibited cytochrome c oxidase purified from bovine heart mitochondria, both in the soluble state and reconstituted in liposomes, are presented. In the soluble oxidase, the H(+)/heme a, Cu(A) ratios were experimentally determined upon oxidation by ferricyanide of these centers as well as upon their reduction by hexammineruthenium(II). These measurements showed that in order to obtain H(+)/heme a, Cu(A) ratios approaching 1, one-step full oxidation of both metal centers by ferricyanide had to be induced by a stoicheiometric amount of the oxidant. Partial stepwise oxidation or reduction of heme a and Cu(A) did produce H(+)/heme a, Cu(A) ratios significantly lower or higher than 1, respectively. The experimental H(+)/heme a, Cu(A) ratios measured upon stepwise reduction/oxidation of the metals were reproduced by mathematical simulation based on the coupling of oxido-reduction of both heme a and Cu(A) to pK shifts of common acid-base groups. The vectorial nature of the proton-electron coupling at heme a/Cu(A) was analyzed by measuring pH changes in the external bulk phase associated with oxido-reduction of these redox centers in the CO-inhibited oxidase reconstituted in liposomes. The results show that the proton release associated with the oxidation of heme a and Cu(A) takes place in the external aqueous phase. Protons taken up by the oxidase upon rereduction of the centers derive, on the other hand, from the inner space. These results provide evidence supporting the view that cooperative proton-electron coupling at heme a/Cu(A) is involved in the proton pump of the oxidase.

Animals↗
PubMed2000

Coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in cytochrome c oxidase. Studies with the carbon monoxide inhibited enzyme.

A study is presented on the coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in carbon monoxide inhibited cytochrome c oxidase isolated from bovine heart mitochondria. Detailed analysis of the coupling number for H(+) release per heme a, Cu(A) oxidized (H(+)/heme a, Cu(A) ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the CO-inhibited fully reduced COX, the equivalents of heme a, Cu(A), and added cytochrome c oxidized and the H(+) released upon oxidation and all taken up back by the oxidase upon rereduction of the metal centers. One of two reductants was used, either succinate plus a trace of mitochondrial membranes (providing a source of succinate-c reductase) or hexaammineruthenium(II) as the chloride salt. The experimental H(+)/heme a, Cu(A) ratios varied between 0.65 and 0.90 in the pH range 6.0-8.5. The pH dependence of the H(+)/heme a, Cu(A) ratios could be best-fitted by a function involving two redox-linked acid-base groups with pK(o)-pK(r) of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that Cu(A) and heme a exhibited superimposed E'(m) values, which decreased, for both metals, by around 20 mV/pH unit increase in the range 6.0-8.5. A model in which oxido-reduction of heme a and Cu(A) are both linked to the pK shifts of the two acid-base groups, characterized by the analysis of the pH dependence of the H(+)/heme a, Cu(A) ratios, provided a satisfactory fit for the pH dependence of the E'(m) of heme a and Cu(A). The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme a and Cu(A) in the proton-pumping activity of cytochrome c oxidase.

Animals↗
PubMed1999

Effects of site-directed mutagenesis of protolytic residues in subunit I of Bacillus subtilis aa3-600 quinol oxidase. Role of lysine 304 in proton translocation.

Various protolytic residues in subunit I of aa3-600 quinol oxidase of the aerobic Gram-positive Bacillus subtilis were mutagenized to nonpolar residues. Two of the mutations, Y284F and K304L, impaired the bioenergetic function of the microorganism. The Y284F mutation suppressed the electron-transfer activity of quinol oxidase and altered its interaction with CO and H2O2, thus showing destruction of the binuclear domain as observed for the bo3 quinol oxidase of Escherichia coli. The K304L mutation did not alter significantly the redox activity of the oxidase and its interaction with CO and H2O2 but suppressed the proton pumping activity of the enzyme. These results show that the K304 residue, which is invariantly conserved (as K or R) in practically all the sequences of the heme-copper oxidases so far available (around 100), is essential for the proton pumping activity of the oxidase.

Amino Acid Sequence↗
PubMed1998

A cooperative model for protonmotive heme-copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase.

Oxido-reductions of metal centers in cytochrome c oxidase are linked to pK shifts of acidic groups in the enzyme (redox Bohr effects). The linkage at heme a results in proton uptake from the inner space upon reduction and proton release in the external space upon oxidation of the metal. The relationship of this process to the features of the proton pump in cytochrome c oxidase and its atomic structure revealed by X-ray crystallography to 2.8-2.3 A resolution is examined. A mechanism for the proton pump of cytochrome c oxidase, based on cooperative coupling at heme a, is proposed.

Amino Acid Sequence↗
PubMed1998

Ferrocyanide-peroxidase activity of cytochrome c oxidase

Redox interaction of mitochondrial cytochrome c oxidase (COX) with ferrocyanide/ferricyanide couple is greatly accelerated by polycations, such as poly-l-lysine [Musatov et al. (1991) Biological Membranes 8, 229-234]. This has allowed us to study ferrocyanide oxidation by COX at very high redox potentials of the ferrocyanide/ferricyanide couple either following spectrophotometrically ferricyanide accumulation or measuring proton uptake associated with water formation in the reaction. At low [ferrocyanide]/[ferricyanide] ratios (Eh values around 500 mV) and ambient oxygen concentration, the ferrocyanide-oxidase activity of COX becomes negligibly small as compared to the reaction rate observed with pure ferrocyanide. Oxidation of ferrocyanide under these conditions, is greatly stimulated by H2O2 or ethylhydroperoxide indicating peroxidatic reaction involved. The ferrocyanide-peroxidase activity of COX is strictly polylysine-dependent and is inhibited by heme a3 ligands such as KCN and NaN3. Apparently the reaction involves normal electron pathway, i.e. electron donation through CuA and oxidation via heme a3. The peroxidase reaction shows a pH-dependence similar to that of the cytochrome c oxidase activity of COX. When COX is preequilibrated with excess H2O2, addition of ferrocyanide shifts the initial steady-state concentrations of the Ferryl-Oxo and Peroxy compounds towards approximately 2:1 ratio of the two intermediates. It is suggested that in the peroxidase cycleferrocyanide donates electrons to both P and F intermediates with a comparable efficiency. Isolation of a partial redox activity of COX opens a possibility to study separately proton translocation coupled to the peroxidase half-reaction of the COX reaction cycle. Copyright 1998

Journal Article↗
PubMed1998

Cooperative coupling and role of heme a in the proton pump of heme-copper oxidases.

In the last few years, evidence has accumulated supporting the applicability of the cooperative model of proton pumps in cytochrome systems, vectorial Bohr mechanisms, to heme-copper oxidases. The vectorial Bohr mechanism is based on short- and long-range protonmotive cooperative effects linked to redox transitions of the metal centers. The crystal structure of oxidized and reduced bovine-heart cytochrome c oxidase reveals, upon reduction, the occurrence of long-range conformational changes in subunit I of the oxidase. Analysis of the crystal structure of cytochrome c oxidase shows the existence of hydrogen-bonded networks of amino acid residues which could undergo redox-linked pK shifts resulting in transmembrane proton translocation. Our group has identified four proteolytic groups undergoing reversible redox-linked pK shifts. Two groups result in being linked to redox transitions of heme a3. One group is apparently linked to CuB. The fourth group is linked to oxido-reduction of heme a. We have shown that the proton transfer resulting from the redox Bohr effects linked to heme a and CuB in the bovine oxidase displays membrane vectorial asymmetry, i.e., protons are taken up from the inner aqueous space (N), upon reduction, and released in the external space (P), upon oxidation of the metals. This direction of proton uptake and release is just what is expected from the vectorial Bohr mechanism. The group linked to heme a, which can transfer up to 0.9 H+/e- at pHs around neutrality, can provide the major contribution to the proton pump. It is proposed that translocation of pumped protons, linked to electron flow through heme a, utilizes a channel (channel D) which extends from a conserved aspartate at the N entrance to a conserved glutamate located between heme a and the binuclear center. The carboxylic group of this glutamic acid, after having delivered, upon electron flow through heme a, pumped protons towards the P phase, once reprotonated from the N phase, moves to deliver, subsequently, to the binuclear center chemical protons consumed in the conversion of the peroxy to ferryl and of the latter to the oxy intermediate in the redox cycle. Site-directed mutagenesis of protolytic residues in subunit I of the aa3-600 quinol oxidase of Bacillus subtilis to non-polar residues revealed that the conserved Lys 304 is critical for the proton pumping activity of the oxidase. Crystal structures of cytochrome c oxidase show that this lysine is at the N entrance of a channel which translocates the protons consumed for the production of the peroxy intermediate. Inhibition of this pathway, by replacement of the lysine, short-circuits protons from channel D to the binuclear center, where they are utilized in the chemistry of oxygen reduction.

Animals↗
PubMed1997

Vectorial nature of redox Bohr effects in bovine heart cytochrome c oxidase.

The vectorial nature of redox Bohr effects (redox-linked pK shifts) in cytochrome c oxidase from bovine heart incorporated in liposomes has been analyzed. The Bohr effects linked to oxido-reduction of heme a and CuB display membrane vectorial asymmetry. This provides evidence for involvement of redox Bohr effects in the proton pump of the oxidase.

Animals↗
PubMed1997

Proton pumping by cytochrome c oxidase is coupled to peroxidase half of its catalytic cycle.

The four-electron reaction cycle of cytochrome oxidase is comprised of an eu-oxidase phase in which the enzyme receives the first two electrons and reduces oxygen to bound peroxide and a peroxidase phase in which the peroxy state formed in the eu-oxidase half of the cycle is reduced by the 3rd and 4th electrons to the ferryl-oxo state and oxidized form, respectively. Here we show that the ferrocyanide-peroxidase activity of cytochrome c oxidase incorporated in phospholipid vesicles is coupled to proton pumping. The H+/e- ratio for the ferrocyanide-peroxidase partial reaction is twice higher than for the overall ferrocyanide-oxidase activity and is close to 2. These results show that proton pumping by COX is confined to the peroxidase part of the enzyme catalytic cycle (transfer of the 3rd and 4th electron) whereas the eu-oxidase part (transfer of the first two electrons) may not be proton pumping.

Animals↗
PubMed1997

Redox-linked protolytic reactions in soluble cytochrome-c oxidase from beef-heart mitochondria: redox Bohr effects.

A study is presented of co-operative redox-linked protolytic reactions (redox Bohr effects) in soluble cytochrome-c oxidase purified from bovine-heart mitochondria. Bohr effects were analyzed by direct measurement, with accurate spectrophotometric and potentiometric methods, of H+ uptake and release by the oxidase associated with reduction and oxidation of hemes a and a3. CuA and CuB in the unliganded and in the CN- or CO-liganded enzyme. The results show that there are in the bovine oxidase four protolytic groups undergoing reversible pK shifts upon oxido-reduction of the electron transfer metals. Two groups with pKox and pKred values around 7 and > 12 respectively appear to be linked to redox transitions of heme a3. One group with pKox and pKred around 6 and 7 is apparently linked to CuB, a fourth one with pKox and pKred of 6 and 9 appears to be linked to heme a. The possible nature of the amino acids involved in the redox Bohr effects and their role in H+ translocation is discussed.

Animals↗
PubMed1997

A possible role of slips in cytochrome C oxidase in the antioxygen defense system of the cell.

Evidence is available showing that the coupling efficiency of the proton pump in cytochrome c oxidase of mitochondria can under certain conditions decrease significantly below the maximum attainable value. The view is developed that slips in the proton pump of cytochrome c oxidase represent an intrinsic switch mechanism which regulates the relative contribution of energy transfer and respiratory protection against oxygen toxicity by the oxidase.

Electron Transport↗
PubMed1996

Factors affecting the H+/e- stoichiometry in mitochondrial cytochrome c oxidase: influence of the rate of electron flow and transmembrane delta pH.

A study is presented of the factors affecting the H+/e- stoichiometry of the proton pump of mitochondrial cytochrome c oxidase, isolated and reconstituted in phospholipid vesicles (COV). Under level flow conditions, i.e., in the absence of a transmembrane delta muH+, the H+/e- ratio, obtained from spectrophotometric measurements of the initial rates of electron flow and H+ release specifically elicited by cytochrome c, varied from around 0 to 1, depending on the actual rate of electron flow through the oxidase. At steady state the H+/e- ratio for the oxidase was specifically depressed by the transmembrane delta pH. The study of the H+/e- ratio of the pump was complemented by an analysis of the redox pattern of cytochrome c, CuA, and heme a. From both sets of results and recent structural data from other groups, it is concluded that the dependence of the H+/e- ratio on the rate of electron flow through the oxidase and transmembrane delta pH is associated with the possible occurrence of two electron transfer pathways in cytochrome c oxidase, a coupled one (cyt c-->CuA-->heme a-->heme a3-CuB) and a decoupled one (cyt c-->CuA-->heme a3-CuB). The contributions of the two pathways, differently affected by kinetics and thermodynamic factors, will determine the actual H+/e- ratio of the pump. A possible role of heme a in the proton pump and the physiological implication of the variable H+/e- ratio in the oxidase are discussed.

Animals↗
PubMed1996

The Saccharomyces cerevisiae OXA1 gene is required for the correct assembly of cytochrome c oxidase and oligomycin-sensitive ATP synthase.

The nuclear gene OXA1 was first isolated in Saccharomyces cerevisiae and found to be required at a post-translational step in cytochrome c oxidase biogenesis, probably at the level of assembly. Mutations in OXA1 lead to a complete respiratory deficiency. The protein Oxa1p is conserved through evolution and a human homolog has been isolated by functional complementation of a yeast oxa1- mutant. In order to further our understanding of the role of Oxa1p, we have constructed two yeast strains in which the OXA1 open reading frame was almost totally deleted. Cytochrome spectra and enzymatic activity measurements show the absence of heme aa3 and of a cytochrome c oxido-reductase activity and dramatic decrease of the oligomycin sensitive ATPase activity. Analysis of the respiratory complexes in non-denaturing gels reveals that Oxa1p is necessary for the correct assembly of the cytochrome c oxidase and the ATP synthase complex.

Adenosine Triphosphatases↗
PubMed1995

Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement.

Using the high efficiency of homologous gene recombination in Bacillus subtilis, a strategy for mutational analysis of the proton pumping aa3-600 quinol oxidase of this organism has been developed. The qox operon with the qoxA, qoxB, qoxC and qoxD genes, coding for the four subunits of this oxidase, was deleted and then replaced with mutated copies in which qoxC (subunit III) or qoxD (subunit IV) genes were deleted. The complete deletion of the qox operon caused disappearance of heme aa3-600 and a slight depression of the overall respiratory activity, compensated by alternative oxidase with no proton pumping activity. Deletion of qoxC probably resulted in a defective assembly of the aa3-600 quinol oxidase. The strain with deletion of qoxD gene expressed normal content of heme aa3-600 but exhibited a reduced respiratory activity and a significantly depressed proton pumping activity. These results show that subunit IV is critical for the activity of the proton pumping aa3-600 quinol oxidase.

Bacillus subtilis↗
PubMed1995

Proton/electron stoichiometry of mitochondrial bc1 complex. Influence of pH and transmembrane delta pH.

The effect of pH and transmembrane delta pH on the efficiency of the proton pump of the mitochondrial bc1 complex both in situ and in the reconstituted state was studied. In both cases the H+/e- ratio for vectorial proton translocation by the bc1 complex respiring at the steady state, under conditions in which the transmembrane pH difference (delta pH) represents the only component of the proton motive force (delta p), was significantly lower than that measured under level flow conditions. The latter amounts, at neutral pH, to 1 (2 including the scalar H+ release). In the reconstituted system steady-state delta pH was modulated by changing the intravesicular buffer as well as the intra/extra-liposomal pH. Under these conditions the H+/e- ratio varied inversely with the delta pH. The data presented show that delta pH exerts a critical control on the proton pump of the bc1 complex. Increasing the external pH above neutrality caused a decrease of the level flow H+/e- ratio. This effect is explained in terms of proton/electron linkage in b cytochromes.

Animals↗
PubMed1994

Role of nuclear-encoded subunits of mitochondrial cytochrome c oxidase in proton pumping revealed by limited enzymatic proteolysis.

The role of supernumerary subunits of bovine heart cytochrome c oxidase has been investigated by examining the effect on the enzymatic activity of limited proteolysis by chymotrypsin, thermolysin, and trypsin. All three proteases, when added to the soluble oxidase, digested subunits III, VIa, and VIb and caused inhibition of electron flow in the oxidase. In addition, trypsin and thermolysin also digested subunit IV. Trypsin cleaved off an N-terminal segment of seven residues; thermolysin cleaved only the first four residues at the N-terminus of subunit IV. Digestion of the soluble oxidase by trypsin but not by thermolysin caused decoupling of redox-linked proton pumping in the oxidase. It is concluded that the sequence V5-V6-K7 of the hydrophilic N-terminus of subunit IV, which protrudes out of the matrix side of the mitochondrial membrane, mediates the access of protons into the transmembrane proton translocating pathway in the oxidase.

Amino Acid Sequence↗
PubMed1994

The proton pump of heme-copper oxidases.

Proton pumping heme-copper oxidases represent the terminal, energy-transfer enzymes of respiratory chains in prokaryotes and eukaryotes. The CuB-heme a3 (or heme o) binuclear center, associated with the largest subunit I of cytochrome c and quinol oxidases, is directly involved in the coupling between dioxygen reduction and proton pumping. The role of the other subunits is less clear. The following aspects will be covered in this paper: i) the efficiency of coupling in the mitochondrial aa3 cytochrome c oxidase. In particular, the effect of respiratory rate and protonmotive force on the H+/e- stoichiometry and the role of subunit IV; ii) mutational analysis of the aa3 quinol oxidase of Bacillus subtilis addressed to the role of subunit III, subunit IV and specific residues in subunit I; iii) possible models of the protonmotive catalytic cycle at the binuclear center. The observations available suggest that H+/e- coupling is based on the combination of protonmotive redox catalysis at the binuclear center and co-operative proton transfer in the protein.

Animals↗