Mitochondrial sAC-cAMP-PKA Axis Modulates the ΔΨm-Dependent Control Coefficients of the Respiratory Chain Complexes: Evidence of Respirasome Plasticity.

The current view of the mitochondrial respiratory chain complexes I, III and IV foresees the occurrence of their assembly in supercomplexes, providing additional functional properties when compared with randomly colliding isolated complexes. According to the plasticity model, the two structural states of the respiratory chain may interconvert, influenced by the intracellular prevailing conditions. In previous studies, we suggested the mitochondrial membrane potential as a factor for controlling their dynamic balance. Here, we investigated if and how the cAMP/PKA-mediated signalling influences the aggregation state of the respiratory complexes. An analysis of the inhibitory titration profiles of the endogenous oxygen consumption rates in intact HepG2 cells with specific inhibitors of the respiratory complexes was performed to quantify, in the framework of the metabolic flux theory, the corresponding control coefficients. The attained results, pharmacologically inhibiting either PKA or sAC, indicated that the reversible phosphorylation of the respiratory chain complexes/supercomplexes influenced their assembly state in response to the membrane potential. This conclusion was supported by the scrutiny of the available structure of the CI/CIII2/CIV respirasome, enabling us to map several PKA-targeted serine residues exposed to the matrix side of the complexes I, III and IV at the contact interfaces of the three complexes.

Bioenergetic profile and redox tone modulate in vitro osteogenesis of human dental pulp stem cells: new perspectives for bone regeneration and repair.

BACKGROUND: Redox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling. METHODS: hDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs. RESULTS: In differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction. CONCLUSIONS: Altogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair.

The allosteric protein interactions in the proton-motive function of mammalian redox enzymes of the respiratory chain.

Insight into mammalian respiratory complexes defines the role of allosteric protein interactions in their proton-motive activity. In cytochrome c oxidase (CxIV) conformational change of subunit I, caused by O2 binding to heme a32+-CuB+ and reduction, and stereochemical transitions coupled to oxidation/reduction of heme a and CuA, combined with electrostatic effects, determine the proton pumping activity. In ubiquinone-cytochrome c oxidoreductase (CxIII) conformational movement of Fe-S protein between cytochromes b and c1 is the key element of the proton-motive activity. In NADH-ubiquinone oxidoreductase (CxI) ubiquinone binding and reduction result in conformational changes of subunits in the quinone reaction structure which initiate proton pumping.

Allosteric Cooperativity in Proton Energy Conversion in A1-Type Cytochrome c Oxidase.

Cytochrome c oxidase (CcO), the CuA, heme a, heme a3, CuB enzyme of respiratory chain, converts the free energy released by aerobic cytochrome c oxidation into a membrane electrochemical proton gradient (ΔµH+). ΔµH+ derives from the membrane anisotropic arrangement of dioxygen reduction to two water molecules and transmembrane proton pumping from a negative (N) space to a positive (P) space separated by the membrane. Spectroscopic, potentiometric, and X-ray crystallographic analyses characterize allosteric cooperativity of dioxygen binding and reduction with protonmotive conformational states of CcO. These studies show that allosteric cooperativity stabilizes the favorable conformational state for conversion of redox energy into a transmembrane ΔµH+.

The mechanism of coupling between oxido-reduction and proton translocation in respiratory chain enzymes.

The respiratory chain of mitochondria and bacteria is made up of a set of membrane-associated enzyme complexes which catalyse sequential, stepwise transfer of reducing equivalents from substrates to oxygen and convert redox energy into a transmembrane protonmotive force (PMF) by proton translocation from a negative (N) to a positive (P) aqueous phase separated by the coupling membrane. There are three basic mechanisms by which a membrane-associated redox enzyme can generate a PMF. These are membrane anisotropic arrangement of the primary redox catalysis with: (i) vectorial electron transfer by redox metal centres from the P to the N side of the membrane; (ii) hydrogen transfer by movement of quinones across the membrane, from a reduction site at the N side to an oxidation site at the P side; (iii) a different type of mechanism based on co-operative allosteric linkage between electron transfer at the metal redox centres and transmembrane electrogenic proton translocation by apoproteins. The results of advanced experimental and theoretical analyses and in particular X-ray crystallography show that these three mechanisms contribute differently to the protonmotive activity of cytochrome c oxidase, ubiquinone-cytochrome c oxidoreductase and NADH-ubiquinone oxidoreductase of the respiratory chain. This review considers the main features, recent experimental advances and still unresolved problems in the molecular/atomic mechanism of coupling between the transfer of reducing equivalents and proton translocation in these three protonmotive redox complexes.

MALDI-TOF MS lipid profiles of cytochrome c oxidases: cardiolipin is not an essential component of the Paracoccus denitrificans oxidase.

Lipids of cytochrome c oxidase (COX) of Paracoccus denitrificans have been identified by MALDI-TOF MS direct analyses of isolated protein complexes, avoiding steps of lipid extraction or chromatographic separation. Two different COX preparations have been considered in this study: the enzyme core consisting of subunits I and II (COX 2-SU) and the complete complex comprising all four subunits (COX 4-SU). In addition, MALDI-TOF MS lipid profiles of bacterial COX are also compared with those of the isolated mitochondrial COX and bacterial bc1 complex. We show that the main lipids associated with bacterial COX 4-SU are phosphatidylglycerol (PG) and phosphatidylcholine (PC), and minor amounts of cardiolipin (CL). PG and PC are absent in the COX 2-SU preparation lacking subunits III and IV, whereas CL is still present. Quantitative analyses indicate that at variance from mitochondrial COX, cardiolipin is present in substoichiometric amounts in bacterial COX, at a CL:COX molar ratio of ∼1:10. We conclude that bacterial COX does not require CL for structure or its activity.

Intramitochondrial adenylyl cyclase controls the turnover of nuclear-encoded subunits and activity of mammalian complex I of the respiratory chain.

In mammalian cells the nuclear-encoded subunits of complex I are imported into mitochondria, where they are assembled with mt-DNA encoded subunits in the complex, or exchanged with pre-existing copies in the complex. The present work shows that in fibroblast cultures inhibition by KH7 of cAMP production in the mitochondrial matrix by soluble adenylyl cyclase (sAC) results in decreased amounts of free non-incorporated nuclear-encoded NDUFS4, NDUFV2 and NDUFA9 subunits of the catalytic moiety and inhibition of the activity of complex I. Addition of permeant 8-Br-cAMP prevents this effect of KH7. KH7 inhibits accumulation in isolated rat-liver mitochondria and incorporation in complex I of "in vitro" produced, radiolabeled NDUFS4 and NDUFV2 subunits. 8-Br-cAMP prevents also this effect of KH7. Use of protease inhibitors shows that intramitochondrial cAMP exerts this positive effect on complex I by preventing digestion of nuclear-encoded subunits by mitochondrial protease(s), whose activity is promoted by KH7 and H89, an inhibitor of PKA.

The oxidative phosphorylation system in mammalian mitochondria.

The chapter provides a review of the state of art of the oxidative phosphorylation system in mammalian mitochondria. The sections of the paper deal with: (i) the respiratory chain as a whole: redox centers of the chain and protonic coupling in oxidative phosphorylation (ii) atomic structure and functional mechanism of protonmotive complexes I, III, IV and V of the oxidative phosphorylation system (iii) biogenesis of oxidative phosphorylation complexes: mitochondrial import of nuclear encoded subunits, assembly of oxidative phosphorylation complexes, transcriptional factors controlling biogenesis of the complexes. This advanced knowledge of the structure, functional mechanism and biogenesis of the oxidative phosphorylation system provides a background to understand the pathological impact of genetic and acquired dysfunctions of mitochondrial oxidative phosphorylation.

The light-activated proton pump Bop I of the archaeon Haloquadratum walsbyi.

We have isolated and characterized the light-driven proton pump Bop I from the ultrathin square archaeon Haloquadratum walsbyi, the most abundant component of the dense microbial community inhabiting hypersaline environments. The disruption of cells by hypo-osmotic shock yielded Bop I retinal protein highly enriched membranes, which contain one main 27 kDa protein band together with a high content of the carotenoid bacterioruberin. Light-induced pH changes were observed in suspensions of Bop I retinal protein-enriched membranes under sustained illumination. Solubilization of H. walsbyi cells with Triton X-100, followed by phenyl-Sepharose chromatography, resulted in isolation of two purified Bop I retinal protein bands; mass spectrometry analysis revealed that the Bop I was present as only protein in both the bands. The study of light/dark adaptations, M-decay kinetics, responses to titration with alkali in the dark and endogenous lipid compositions of the two Bop I retinal protein bands showed functional differences that could be attributed to different protein aggregation states. Proton-pumping activity of Bop I during the photocycle was observed in liposomes constituted of archaeal lipids. Similarities and differences of Bop I with other archaeal proton-pumping retinal proteins will be discussed.

Allosteric interactions and proton conducting pathways in proton pumping aa(3) oxidases: heme a as a key coupling element.

In this paper allosteric interactions in protonmotive heme aa(3) terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H(+)/e(-) coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa(3) oxidase, which decreases by more than 200mV the E(m) of heme a, inhibits proton pumping. Mutational amino acid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa(3) oxidases, as well as Zn(2+) binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O(2) to 2 H(2)O.

Inhibition of proton pumping in membrane reconstituted bovine heart cytochrome c oxidase by zinc binding at the inner matrix side.

A study is presented on the effect of zinc binding at the matrix side, on the proton pump of purified liposome reconstituted bovine heart cytochrome c oxidase (COV). Internally trapped Zn(2+) resulted in 50% decoupling of the proton pump at level flow. Analysis of the pH dependence of inhibition by internal Zn(2+) of proton release in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase indicates that Zn(2+) suppresses two of the four proton pumping steps in the cycle, those taking place when the 2 OH(-) produced in the reduction of O(2) at the binuclear center are protonated to 2 H(2)O. This decoupling effect could be associated with Zn(2+) induced conformational alteration of an acid/base cluster linked to heme a(3).

Redox Bohr effects and the role of heme a in the proton pump of bovine heart cytochrome c oxidase.

Structural and functional observations are reviewed which provide evidence for a central role of redox Bohr effect linked to the low-spin heme a in the proton pump of bovine heart cytochrome c oxidase. Data on the membrane sidedness of Bohr protons linked to anaerobic oxido-reduction of the individual metal centers in the liposome reconstituted oxidase are analysed. Redox Bohr protons coupled to anaerobic oxido-reduction of heme a (and Cu(A)) and Cu(B) exhibit membrane vectoriality, i.e. protons are taken up from the inner space upon reduction of these centers and released in the outer space upon their oxidation. Redox Bohr protons coupled to anaerobic oxido-reduction of heme a(3) do not, on the contrary, exhibit vectorial nature: protons are exchanged only with the outer space. A model of the proton pump of the oxidase, in which redox Bohr protons linked to the low-spin heme a play a central role, is described. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

The inhibitory binding site(s) of Zn2+ in cytochrome c oxidase.

EXAFS analysis of Zn binding site(s) in bovine-heart cytochrome c oxidase and characterization of the inhibitory effect of internal zinc on respiratory activity and proton pumping of the liposome reconstituted oxidase are presented. EXAFS identifies tetrahedral coordination site(s) for Zn(2+) with two N-histidine imidazoles, one N-histidine imidazol or N-lysine and one O-COOH (glutamate or aspartate), possibly located at the entry site of the proton conducting D pathway in the oxidase and involved in inhibition of the oxygen reduction catalysis and proton pumping by internally trapped zinc.

Concerted involvement of cooperative proton-electron linkage and water production in the proton pump of cytochrome c oxidase.

In cytochrome c oxidase, oxido-reductions of heme a/Cu(A) and heme a3/Cu(B) are cooperatively linked to proton transfer at acid/base groups in the enzyme. H+/e- cooperative linkage at Fe(a3)/Cu(B) is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H+/e- linkage at heme a (and Cu(A)). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H2O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu(A) and heme a3/Cu(B) in the soluble oxidase; (iii) H+ release in the external phase (i.e. H+ pumping) associated with the oxidative (R-->O transition), reductive (O-->R transition) and a full catalytic cycle (R-->O-->R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H+/e- linkage at heme a/Cu(A) and heme a3/Cu(B) with acid/base clusters, C1 and C2 respectively, and protonmotive steps of the reduction of O2 to water are involved in proton pumping.

pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase. The role of H2O produced at the oxygen-reduction site.

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.

Protonmotive cooperativity in cytochrome c oxidase.

Cooperative linkage of solute binding at separate binding sites in allosteric proteins is an important functional attribute of soluble and membrane bound hemoproteins. Analysis of proton/electron coupling at the four redox centers, i.e. Cu(A), heme a, heme a(3) and Cu(B), in the purified bovine cytochrome c oxidase in the unliganded, CO-liganded and CN-liganded states is presented. These studies are based on direct measurement of scalar proton translocation associated with oxido-reduction of the metal centers and pH dependence of the midpoint potential of the redox centers. Heme a (and Cu(A)) exhibits a cooperative proton/electron linkage (Bohr effect). Bohr effect seems also to be associated with the oxygen-reduction chemistry at the heme a(3)-Cu(B) binuclear center. Data on electron transfer in cytochrome c oxidase are also presented, which, together with structural data, provide evidence showing the occurrence of direct electron transfer from Cu(A) to the binuclear center in addition to electron transfer via heme a. A survey of structural and functional data showing the essential role of cooperative proton/electron linkage at heme a in the proton pump of cytochrome c oxidase is presented. On the basis of this and related functional and structural information, variants for cooperative mechanisms in the proton pump of the oxidase are examined.

A cooperative model for proton pumping in cytochrome c oxidase.

In this paper, the mechanism of proton pumping in cytochrome c oxidase is examined. Data on cooperative linkage of vectorial proton translocation to oxido-reduction of Cu(A) and heme a in the CO-inhibited, liposome-reconstituted bovine cytochrome c oxidase are reviewed. Results on proton translocation associated to single-turnover oxido-reduction of the four metal centers in the unliganded, membrane-reconstituted oxidase are also presented. On the basis of these results, X-ray crystallographic structures and spectrometric data for a proton pumping model in cytochrome c oxidase is proposed. This model, which is specifically derived from data available for the bovine cytochrome c oxidase, is intended to illustrate the essential features of cooperative coupling of proton translocation at the low potential redox site. Variants will have to be introduced for those members of the heme copper oxidase family which differ in the redox components of the low potential site and in the amino acid network connected to this site. The model we present describes in detail steps of cooperative coupling of proton pumping at the low potential Cu(A)-heme a site in the bovine enzyme. It is then outlined how this cooperative proton transfer can be thermodynamically and kinetically coupled to the chemistry of oxygen reduction to water at the high potential Cu(B)-heme a(3) center, so as to result in proton pumping, in the turning-over enzyme, against a transmembrane electrochemical proton gradient of some 250 mV.

Proton transfer reactions associated with the reaction of the fully reduced, purified cytochrome C oxidase with molecular oxygen and ferricyanide.

A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from approximately 3.0 H(+)/COX at pH 6.2-6.3 to approximately 1.2 H(+)/COX at pH 8.0-8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from approximately 1.0 H(+)/COX at pH 6.2-6.3 to approximately 2.8 H(+)/COX at pH 8.0-8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3-1.6 H(+)/COX in the pH range 6.2-8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O(2) to 2H(2)O. The results show that the expected stoichiometric proton consumption of 4H(+) in the reduction of O(2) to 2H(2)O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H(+)/COX are initially taken up in the reduction of O(2) to two OH(-) groups bound to the binuclear Fe a(3)-Cu(B) center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H(2)O. At alkaline pHs the third and fourth protons are taken up with formation of 2H(2)O only upon rereduction of COX.