Isoforms of the DHTKD1-Encoded 2-Oxoadipate Dehydrogenase, Identified in Animal Tissues, Are not Observed upon the Human DHTKD1 Expression in Bacterial or Yeast Systems.

Unlike the OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH), which is an essential enzyme present in all animal tissues, expression of the DHTKD1-encoded isoenzyme, 2-oxoadipate dehydrogenase (OADH), depends on a number of factors, and mutant DHTKD1 phenotypes are rarely manifested. Physiological significance of OADH is also obscured by the fact that both isoenzymes transform 2-oxoglutarate and 2-oxoadipate. By analogy with other members of the 2-oxo acid dehydrogenases family, OADH is assumed to be a component of the multienzyme complex that catalyzes oxidative decarboxylation of 2-oxoadipate. This study aims at molecular characterization of OADH from animal tissues. Phylogenetic analysis of 2-oxo acid dehydrogenases reveals OADH only in animals and Dictyostelium discoideum slime mold, within a common branch with bacterial OGDH. Examination of partially purified animal OADH by immunoblotting and mass spectrometry identifies two OADH isoforms with molecular weights of about 130 and 70 kDa. These isoforms are not observed upon the expression of human DHTKD1 protein in either bacterial or yeast system, where the synthesized OADH is of expected molecular weight (about 100 kDa). Thus, the OADH isoforms present in animal tissues, may result from the animal-specific regulation of the DHTKD1 expression and/or posttranslational modifications of the encoded protein. Mapping of the peptides identified in the OADH preparations, onto the protein structure suggests that the 70-kDa isoform is truncated at the N-terminus, but retains the active site. Since the N-terminal domain of OGDH is required for the formation of the multienzyme complex, it is possible that the 70-kDa isoform catalyzes non-oxidative transformation of dicarboxylic 2-oxo acids that does not require the multienzyme structure. In this case, the ratio of the OADH isoforms in animal tissues may correspond to the ratio between the oxidative and non-oxidative decarboxylation of 2-oxoadipate.

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.

Cloning of Pichia pastoris Fet3: insights into the high affinity iron uptake system.

High-affinity iron uptake by yeast cells appears to require the presence of a complex formed on the plasma membrane by the multicopper oxidase Fet3 and the permease Ftr1 which work together to allow iron to enter safely inside the cell. The Pichia pastoris ferroxidase Fet3 has been cloned and it has been found to display high sequence similarity to other yeast multicopper oxidases, including all the predicted ligands for the catalytic copper atoms and for the iron substrate. P. pastoris appears to possess a high-affinity iron uptake system similar to that of S. cerevisiae, as far as regulation of expression is concerned. However, the P. pastoris high-affinity iron uptake system presents a K(m) value for iron almost ten times higher than that of S. cerevisiae, possibly to control iron fluxes over a wider range of concentrations of this metal, in order to avoid toxic iron overloading.

A free cysteine residue at the dimer interface decreases conformational stability of Xenopus laevis copper,zinc superoxide dismutase.

The two Cu,Zn superoxide dismutases from the amphibian Xenopus laevis (denoted XSODA and XSODB) display different heat sensitivities, XSODA being more thermolabile than XSODB. In this study, we have investigated the contribution of a free cysteine residue located close to the subunit interface of XSODA to its lower thermal stability. We have found that mutation of residue Cys 150 to Ala in XSODA makes the thermal stability of this enzyme comparable to that of the wild-type XSODB isoenzyme, while the introduction of a cysteine residue in the same position of XSODB renders this enzyme variant much more heat-sensitive. Differential scanning calorimetry experiments showed that XSODA has a melting temperature about 8.5 degrees C lower than that of XSODB. On the contrary, the melting temperature of XSODACys150Ala is very close to that of XSODB, while the melting temperature of XSODBSer150Cys is even lower than that of wild-type XSODA. These data indicate that the free cysteine residue present in XSODA affects not only the reversibility of unfolding of the enzyme but also its conformational stability. We suggest that the large effect of the Cys 150 residue on XSODA stability might be due to incorrect disulfide bond formation or disulfide bond interchange during heat-induced unfolding rather than to alteration of the interaction between the enzyme subunits.

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.

Release of highly active Fet3 from membranes of the yeast Pichia pastoris by limited proteolysis.

A soluble derivative of Fet3 has been obtained from the methylotrophic yeast Pichia pastoris by limited proteolysis of membrane suspensions with trypsin. The soluble protein and the membrane-bound parent Fet3 have been purified to apparent homogeneity. Soluble Fet3 had molecular mass 100 kDa, while the full-length protein had molecular mass 110 kDa, in line with the expected decrease for cleavage and loss of a single transmembrane helix and a small cytoplasmic domain. The optical and EPR spectra of Fet3 were typical of the multicopper oxidases, indicating the presence of one type 1 blue copper site and a type 2/type 3 copper trinuclear cluster. V(max) values for iron oxidation by P. pastoris Fet3 were obtained similar to human ceruloplasmin and much higher than those reported for Saccharomyces cerevisiae Fet3.

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.

Ceruloplasmin impairs endothelium-dependent relaxation of rabbit aorta.

This study evaluated the effects of ceruloplasmin, the copper-containing blue oxidase of vertebrate plasma, on the relaxation of rabbit aortic rings after endothelial release of nitric oxide (NO). Ceruloplasmin at physiological, i.e., micromolar, concentrations inhibited relaxation of rabbit aorta induced by endothelium-dependent agonists like acetylcholine or ADP, whereas it was ineffective toward vasodilation due to direct stimulation of smooth muscle cells by nitroglycerin. The effect was reversible and specific for native, fully metalated ceruloplasmin, since relaxation was not impaired by the heat-treated or metal-depleted derivatives. A trapping mechanism, involving a direct interaction of NO or other NO-containing species (like nitrosothiols and iron-dinitrosyls) with the copper sites and/or with the free thiol of ceruloplasmin, could be safely excluded on the basis of spectroscopic and chemical analyses of the protein exposed to authentic NO, nitrosothiols, or iron-dinitrosyls. The data presented in this paper constitute the first evidence of impairment of the endothelium-dependent vasodilatation by a plasma protein and may shed some light on the still uncertain physiological role of ceruloplasmin.

Divalent cation binding to ceruloplasmin.

Binding of calcium to human and sheep ceruloplasmin was investigated by metal substitution with manganese and competitive displacement of bound manganese by calcium monitored by electron paramagnetic resonance spectroscopy. The Kd for calcium was found to be 1.4 mM. Magnesium also bound to ceruloplasmin, with Kd = 0.3 and 0.7 mM for the human and sheep protein, respectively. The thermal stability of ceruloplasmin, as studied by differential scanning calorimetry, was affected by calcium but not by magnesium. A considerable increase of the Tm value, from 73.8 to 83.1 degrees C, was observed for sheep ceruloplasmin in the presence of calcium. The Tm value of the human protein was only slightly altered by calcium (from 85.1 to 87 degrees C). The interaction of ceruloplasmin with the chromatographic material used for its isolation, Sepharose 4B derivatized with chloroethylamine, was weakened by calcium. This allowed us to set up a novel purification scheme that made it possible to efficiently isolate ceruloplasmin and prothrombin from plasma with the same single-step chromatography.

Modulation of the redox state of the copper sites of human ceruloplasmin by chloride.

Incubation of human ceruloplasmin with physiological concentrations of chloride at neutral pH invariably caused dramatic changes of both the spectroscopic and the functional properties of the protein. The optical intensity at 610 nm increased up to 60%, with a concomitant decrease at 330 nm and the appearance of new bands between 410 and 500 nm. Signals previously undetectable appeared in the EPR spectrum. On the basis of computer simulations, they were interpreted as stemming from an oxidized type 1 copper site and from a half-reduced type 3 copper pair. Removal of chloride completely restored the original optical and EPR lineshapes. Hydrogen peroxide, added to ceruloplasmin in the presence of chloride, was able to capture the electron of the half-reduced type 3 site and to yield a protein insensitive to subsequent removal and readdition of the anion. As a whole, the spectroscopic data indicate that a blue site is partially reduced in the resting protein and that, upon binding of chloride, human ceruloplasmin undergoes a structural change leading to displacement of an electron from the reduced type 1 site to the type 3 site pair. Chloride dramatically affected the catalytic efficiency of human ceruloplasmin. At neutral pH, the anion was an activator of the oxidase activity, being able to enhance up to tenfold the catalytic rate. At pH < 6, in line with all previous reports, chloride strongly inhibited the activity. At intermediate pH values, i.e., around 6, the effect was composite, with an activating effect at low concentration and an inhibitory effect at higher concentration. Since chloride is present at very high concentrations in the plasma, these results suggest that human ceruloplasmin is, in the plasma, under control of this anion.

The state of the copper sites in human ceruloplasmin.

The state of the various, spectroscopically distinguishable copper sites (type 1, type 2, and type 3 copper) of human ceruloplasmin was investigated by electron spin resonance (ESR) spectroscopy. The ESR measurements were performed at 100 K and at X-band during the reaction of the protein with either ascorbate or with ferricyanide. A method was developed to directly measure the contribution of type 1 and type 2 copper signals to the ESR spectrum of the native protein. A signal arising from an unperturbed type 2 copper site, obtained by aerobically treating the protein with ascorbate, allowed the estimation that the number of type 2 copper centers detectable by ESR was substantially lower than unity. A fraction of type 1 copper sites was found to be in the reduced state and could be reoxidized by treatment with ferricyanide. The data obtained were consistent with the presence of three type 1 copper sites per protein molecule. Based on the experimentally determined stoichiometries, computer simulations of the ESR lineshape were carried out which confirmed the presence of three nonequivalent type 1 copper sites and of a noninteger amount of ESR-detectable type 2 copper in human ceruloplasmin.

Age-related changes in human ceruloplasmin. Evidence for oxidative modifications.

Human plasma or serum from donors of age comprised between 15 and 95 years was analyzed for paramagnetic and total copper content, as well as for immunoreactive ceruloplasmin content and oxidase activity. All parameters were essentially unaltered, except the paramagnetic copper content, which increased 2-fold upon aging. A dramatic change of the electron paramagnetic resonance spectrum due to ceruloplasmin occurred in individuals over 65 years old and was associated with both an increase of the type 1 copper signal intensity and the appearance of new resonances of a type 2 copper species. Ceruloplasmin was isolated from either young or old donors. Spectroscopic analyses of the isolated proteins confirmed the tendency of type 1 copper to stay reduced in the "young" and oxidized in the "old" protein. The type 2 copper signal observed in most young ceruloplasmin samples was different from the species invariably present in the old protein. The magnetic parameters of the latter species were more consistent with a partially reduced trinuclear copper site. In vitro limited proteolysis resulted in identical fragmentation patterns and kinetics in both proteins. However, changes of the net electric charge were detected in the fragments of the protein isolated from aged individuals, which exhibited a carbonyl content of 0.6 mol of carbonyl/mol of protein. The same pattern of modifications, including a higher carbonyl content (0.65 versus 0.2 mol of carbonyl/mol of protein), could be reproduced by exposure of the young protein to the metal-catalyzed oxidation system iron/ascorbate. These results suggest that during aging ceruloplasmin is subjected to oxidative modifications which are likely to be the source of conformational changes around the copper sites leading to an intramolecular electron rearrangement among the various copper sites.

Ceruloplasmin in human plasma and its relationships with the copper-albumin complex.

The electron paramagnetic spectrum of human plasma is dominated, in the g = 2 region, by resonances from copper atoms bound to ceruloplasmin, and does not reveal the fraction of copper normally associated with albumin, except in a few cases, where a copper-albumin signal increases with time after blood withdrawal. This copper-albumin complex is responsible for a resonance at a g value below g = 2 in the spectrum of human serum, which has been recently attributed to a modified form of type 2 copper bound to ceruloplasmin [Rylkov, V.V., Tarasiev, M.Y. & Moshkov, K.A. (1991) Eur. J. Biochem. 197, 185-189]. In the plasma, copper associated to albumin comes from ceruloplasmin. Purified ceruloplasmin is unable to exchange copper with albumin, either purified or in plasma. It can not be ruled out that some serum components trigger the metal exchange, in a defence mechanism operating when ceruloplasmin leaks, by unknown processes, its copper content before discharging the metal into the various organs.

Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin.

1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)

The multidomain structure of ceruloplasmin from calorimetric and limited proteolysis studies.

Differential scanning calorimetry has been used to investigate the thermal stability of three different ceruloplasmins (from sheep, chicken, and turtle) in their native state and after limited proteolysis. The three undegraded proteins showed a similar structural organization in three calorimetric domains, although their temperature of unfolding varied from 57.8 degrees C (turtle) to 71.2 degrees C (sheep) to 82.1 degrees C (chicken). The spectroscopic and the catalytic properties were totally lost at temperatures corresponding to the unfolding of the less thermostable domain in the case of sheep and chicken ceruloplasmins and to the unfolding of the most thermostable domain in the turtle protein. Trypsin, but not plasmin, digestion caused a significant decrease of the thermal stability of sheep and chicken ceruloplasmins. Turtle ceruloplasmin was insensitive to both proteases. Comparing the thermodynamic parameters of the sheep protein in its undegraded and cleaved states revealed a mismatch between the three calorimetric domains and the 3-fold internal replication of the primary structure, which is evident in the highly homologous, fully sequenced human protein. Copper removal caused the rearrangement of the molecule in only two calorimetric domains, suggesting a role of the metal atoms in organizing a new calorimetric domain, which was tentatively assigned to the less thermostable cooperative unit of the native protein.