Amalia Lania

The essential role of Glu-185 and Tyr-354 residues in the ferroxidase activity of Saccharomyces cerevisiae Fet3.

The structural determinants required for ferroxidase activity by the yeast multicopper oxidase Fet3 have been partially clarified by site‐directed mutagenesis based on homology modeling. Glu‐185 and Tyr‐354 were substituted with Ala and Phe, respectively. Fet3 E185A retained ca. 5% residual ferroxidase catalytic efficiency, and almost 40% oxidase efficiency. On the other hand, Fet3 Y354F exhibited 50% residual efficiency as a ferroxidase and more than 70% as an oxidase. These results provide new insights in the mechanism of iron binding and oxidation by Fet3, establishing the essential role of Glu‐185 and Tyr‐354, and allowing to dissect ferroxidase from non‐iron oxidase activity.

Reduced sensitivity of O2 transport to allosteric effectors and temperature in loggerhead sea turtle hemoglobin: functional and spectroscopic study

The functional and spectroscopic (EPR and absorbance) properties of the adult loggerhead sea turtle (Caretta caretta) hemoglobin have been studied with special reference to the action of allosteric effectors and temperature. Present results indicate that turtle Hb displays a very low O2 affinity and a very small sensitivity to allosteric effectors and temperature. Furthermore, the amplitude of the Bohr effect for O2 binding is strongly reduced. In parallel, EPR and absorbance spectroscopic properties of the nitrosylated derivative of turtle Hb suggest that the hemoprotein is in a low-affinity conformation, even in the absence of allosteric effectors. These findings suggest the existence of unusual molecular mechanisms modulating the basic reaction of Hb with O2, which may be linked to specific physiological needs related to the diving behavior of the turtle.

Mutational analysis of the iron binding site of Saccharomyces cerevisiae ferroxidase Fet3. An in vivo study

The role of residues predicted to be involved in the binding of iron by the yeast ferroxidase Fet3 has been studied by site‐directed mutagenesis. The effect of Fet3 mutations E185A, E185Q, Y354F, D409V and H489D has been investigated in vivo by kinetic analyses of high affinity iron uptake. Our results indicate that Glu‐185 is critical for the binding of iron, since substitution of this residue with Ala or Gln strongly affects both growth and the kinetic parameters of high affinity iron uptake, greatly increasing K m. Mutations Y354F and D409V result in less severe alteration of high affinity iron uptake, while mutant H489D is unable to grow under conditions of iron limitation.

CHARACTERIZATION OF CU,ZN SUPEROXIDE DISMUTASE FROM THE BATHOPHILE FISH, LAMPANYCTUS CROCODILUS

Cu,Zn SOD from the bathophile teleost Lampanyctus crocodilus (LSOD) shows a high degree of homology with the sequence of the enzymes from other teleostean fish species. The catalytic properties of LSOD are very similar to those of the bovine enzyme, albeit with higher sensitivity to thermal denaturation. The apparent molecular mass of LSOD (37.6 KDa) is higher than the other Cu,Zn SOD variants studied. The aminoacid sequence of LSOD reveals interesting substitutions compared to the bovine enzyme. These are discussed in view of the particular environmental conditions to which L. crocodilus is adapted.

Unusual stability properties of a reptilian ceruloplasmin

Ceruloplasmin from the turtle Caretta caretta was isolated to purity by using the single-step procedure recently developed by us to purify sheep and chicken ceruloplasmin. It has a Mr of ca. 145,000 and a total copper content of 5.1 +/- 0.2 atoms of copper per molecule, 50% of which are detectable by EPR. The spectroscopic features include an absorption maximum at 603 nm in the electronic spectrum and the total absence of any resonance attributable to Type 2 copper in the EPR spectrum. Turtle ceruloplasmin was found to be unusually resistant to aging and proteolysis, when compared to ceruloplasmins isolated from other species. p-Phenyl-endiamine oxidase activity measurements revealed an unusually low catalytic efficiency, while the kinetic parameters of Fe(II) oxidation were consistent with those reported for other species of ceruloplasmin.

Isolation and characterization of Cu,Zn superoxide dismutase of the shark Prionace glauca

A Cu,Zn superoxide dismutase was purified for the first time from an elasmobranch species (Prionace glauca) and showed the following differences with respect to other animal superoxide dismutases. The enzyme displays a low isoelectric point. The enzyme activity is unusually independent of ionic strength. The isolated enzyme has 30% of its copper in the reduced state.

A comparative study on the anion binding properties of Bos taurus and Prionace glauca Cu,Zn superoxide dismutases native and chemically modified at lysines

Abstract The interaction between the competitive inhibitor azide and ox and shark Cu,Zn Superoxide dismutases (SODs), in the native state and chemically modified at lysines, has been investigated by optical and EPR spectroscopy. The affinity constants for the anion of the native proteins are identical whilst that of the modified shark SOD is 23% greater with respect to that of the modified bovine enzyme. The difference is attributed to the shark SODs Arg134 residue, a lysine in the bovine enzyme, confirming that this charge location makes a large contribution in steering anions towards the copper active site.