Proteomic analysis of human nasal mucosa: different expression profile in rhino-pathologic states.

BACKGROUND: Rhinitis comprises several diseases with varying causes and different clinical manifestations and pathological features, but treated as a single clinical disorder. As heterogeneous disease, proper differential diagnosis is useful to delineate appropriate therapeutic intervention. Comparative proteomic investigation was aimed to provide information for specific differentially expressed proteins in rhino pathologic state, that could be used for diagnostic purpose and therapeutic monitoring. METHODS: Proteins extracted from nasal mucosa cells of patients with different features of rhinitis and from control subjects, were separated by 2-DE. Proteins differentially expressed were identified by mass spectrometry (MS). RESULTS: Comparative proteomic analyses led to the identification of eighteen proteins differentially expressed in patients with rhinitis, mainly related to cell defense and innate and acquired immunity. From that, at least one protein can be a possible candidate as biomarker of disease.

Morphological and biochemical characterization of mitochondria in Torpedo red blood cells.

A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.

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.

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.

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.

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.

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.

H+/e- stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes. Rate-dependent changes of the stoichiometry in the cytochrome c oxidase vesicles.

The H+/e- stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e- flow and H+ translocation. It is shown that the ----H+/e- ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.

The cytochrome chain of mitochondria exhibits variable H+/e- stoichiometry.

A study is presented of the ----H+/e- stoichiometry for H+ pumping by the cytochrome chain in isolated rat liver mitochondria under level-flow and steady-state conditions. It is shown that the ----H+/e- stoichiometry for the cytochrome chain varies under the influence of the flow rate and transmembrane delta microH+. The rate-dependence is shown to be associated with cytochrome c oxidase, whose ----H+/e- ratio varies from 0 to 1, whilst the ----H+/e- ratio for the span covered by cytochrome c reductase is invariably 2.

Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme.

This paper contributes to the characterization of partial steps of electron and proton transfer in mitochondrial cytochrome c oxidase with respect to their membrane arrangement and involvement in energy-linked protonmotive activity. It is shown that delta psi controls electron flow from cytochrome c to heme a is consistent with the view that the latter center is buried in the membrane in a central position. The pressure exerted by delta psi on oxidation of heme alpha 3 by O2 indicates also that this center is buried in the membrane at some distance from the inner side and is consistent with observations showing that protons consumed in the reduction of O2 to H2O derive from the inner space. Electron flow from heme alpha to heme alpha 3 is shown to be specifically controlled by delta pH and in particular by the pH of the inner phase. Analysis of the effect of DCCD treatment of oxidase vesicles reveals that concentrations of this reagent which result in selective modification of subunit III (Prochaska et al., 1981) produce inhibition of redox-linked proton release. Higher concentrations of DCCD which result also in modification of subunits II and IV (Prochaska et al., 1981) cause inhibition of the pH-dependent electron-transfer step from heme alpha to heme alpha 3.

Role of supernumerary subunits in mitochondrial cytochrome c oxidase.

Role of supernumerary subunits of bovine heart cytochrome c oxidase has been investigated by examining the influence on the enzymatic activity of their removal by chromatographic procedures or controlled digestion by trypsin. Is has been shown that partial proteolytic cleavage of subunit IV results in depression of respiratory activity and of redox-linked proton translocation. Selective removal by gel-filtration of subunit Vlb has no significant influence on the redox and protonmotive activity of the oxidase.

Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes.

In this paper a study is presented of the characteristics of redox-linked proton ejection exhibited by isolated beef-heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented 'right-side out' as in the mitochondrial membrane. The effects on the H+/e- stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K+ to ensure charge compensation produced H+/e- ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H+/e- ratios around 0.3. The H+/e- ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0-7.7, which decreased to negligible values at acidic pH with apparent pKa of 6.7-6.3. Mg2+ and Ca2+ caused a marked depression of the H+/e- ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible. Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox-linked proton ejection in reconstituted cytochrome c oxidase.

Characteristics and nature of redox-linked proton transfer reactions in cytochrome c oxidase of mitochondria.

A discussion is presented of the characteristics of proton transfer reactions associated to redox catalysis in mitochondrial cytochrome c oxidase. These properties are examined in the light of the mechanisms proposed for the conversion of redox energy into a transmembrane proton gradient. It is concluded that this energy transfer process is first of all due to the anisotropic arrangement of the reduction of oxygen to H2O in the oxidase. The experimental observations available seem, on the other hand, to raise doubts on the capacity of cytochrome oxidase to function under steady-state conditions, in the native membrane, as a proton pump.

Characteristics of redox-linked proton ejection in cytochrome c oxidase reconstituted in phospholipid vesicles. New observations support mechanisms different from proton pumping.

Experimental observations reveal a number of characteristics of the redox-linked proton ejection from cytochrome c oxidase vesicles, which apparently cannot be explained by a proton pumping activity of the oxidase. These observations seem, on the other hand, to provide useful elements for alternative explanation(s) of the proton ejection. It is proposed here that the process is scalar and not vectorial and can derive from redox-linked rupture of protonated salt-bridges in the oxidase-lipid complex.