Emanuele De Nitto

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.

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.

Dietary Choline Deprivation Impairs Rat Brain Mitochondrial Function and Behavioral Phenotype

Dietary choline deprivation (CD) is associated with behavioral changes, but mechanisms underlying these detrimental effects are not well characterized. For instance, no literature data are available concerning the CD effects on brain mitochondrial function related to impairment in cognition. Therefore, we investigated brain mitochondrial function and redox status in male Wistar rats fed a CD diet for 28 d. Moreover, the CD behavioral phenotype was characterized. Compared with rats fed a control diet (CTRL), CD rats showed lower NAD-dependent mitochondrial state III and state IV respiration, 40% lower complex I activity, and significantly higher reactive oxygen species production. Total glutathione was oxidatively consumed more in CD than in CTRL rats and the rate of protein oxidation was 40% higher in CD than in CTRL rats, reflecting an oxidative stress condition. The mitochondrial concentrations of cardiolipin, a phospholipid required for optimal activity of complex I, was 20% lower in CD rats than in CTRL rats. Compared with CTRL rats, the behavioral phenotype of CD rats was characterized by impairment in motor coordination and motor learning assessed with the rotarod/accelerod test. Furthermore, compared with CTRL rats, CD rats were less capable of learning the active avoidance task and the number of attempts they made to avoid foot shock was fewer. The results suggest that CD-induced dysfunction in brain mitochondria may be responsible for impairment in cognition and underline that, similar to the liver, the brain also needs an adequate choline supply for its normal functioning.

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

The vectorial nature of redox Bohr effects (redoxlinked 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.

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.

A multidisciplinary study of the extracutaneous pigment system of European sea bass (Dicentrarchus labrax L.). A possible relationship between kidney disease and dopa oxidase activity level

Infectious diseases and breeding conditions can influence fish health status. Furthermore it is well known that human and animal health are strongly correlated. In lower vertebrates melano-macrophage centres, clusters of pigment-containing cells forming the extracutaneous pigment system, are widespread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. Hence, they are employed as biomarker of fish health status. We have investigated this cell system in the European sea bass (Dicentrarchus labrax L.) following the enzyme activities involved in melanin biosynthesis. We have found a possible relationship between kidney disease of farmed fishes and dopa oxidase activity level, suggesting it as an indicator of kidney disease. Moreover variations of dopa oxidase activity in extracutaneous pigment system have been observed with respect to environmental temperature. At last, for the first time, using femtosecond transient absorption spectroscopy (Femto-TA), we pointed out that pigment-containing cells of fish kidney tissue present melanin pigments.

Characteristics of the Protonmotive Activity of the Cytochrome Chain of Mitochondria

In the last years the stoichiometry and mechanism of the protonmotive activity of respiratory chains (1) have become the subject of considerable debate and disagreement (2-9).