Fe3+ binding to ovotransferrin in the presence of alpha-amino acids.

The ability of L-alpha-amino acids as synergistic anions for iron binding to ovotransferrin was investigated through electronic spectroscopy. Glycine and glutamic acid were found to form by far the most stable ternary Fe(3+)-ovotransferrin-amino acid complexes. Less stable adducts were formed by amino acids with a hydroxy, amide or sulphur-containing group in the side chain, while the complexes with leucine, isoleucine, valine, lysine, arginine, tyrosine and tryptophan failed to form. Evidence is obtained that the synergistic effectiveness of the H2N-CH-COO- moiety is determined not only by the isoelectric point of the amino acid and the steric hindrance of its side chain, but a significant role is also played by interactions of the side chain itself with residues in the metal binding domains. Zn2+, Cd2+ and Co2+ are found to bind to ovotransferrin in the presence of glycine. 113Cd-NMR spectra on the Cd-derivative indicate that, according to the interlocking-sites model, the amino group of glycine directly binds to the metal ion.

Redox properties of cytochrome c.

The redox properties of cytochromes (cyt) c, a ubiquitous class of heme-containing electron transport proteins, have been extensively investigated over the last two decades. The reduction potential (E degrees') is central to the chemistry of cyt c for two main reasons. First, E degrees' influences both the thermodynamic and kinetic aspects of the electron exchange reaction with redox partners. Second, this thermodynamic parameter is remarkably sensitive to changes in the properties of the heme and the protein matrix, and hence can be profitably used for the investigation of the solution chemistry of cyt c. This research area owes much to the exploitation of voltammetric techniques for the determination of E degrees' for metalloproteins, which dates back to the late 1970s. Since then, much effort has been devoted to the comprehension of the molecular factors that control E degrees' in cyt c, which include first coordination sphere effects on the heme iron, the interactions of the heme group with the surrounding polypeptide chain and the solvent, and also include medium effects related to the nature and ionic composition of the solvent, pH, the presence of potential protein ligands, and the temperature. This article provides an overview of the most significant advances made in this field recently.

The role of anionic, imidic, and amidic forms in structure-activity relationships. Correlation of electronic indices and bacteriostatic activity in sulfonamides.

The problem of structure-activity relationships in sulfonamide type compounds is tackled on the ground that both bacteriostatic activities and structural indices must be referred to the specific individual forms assumed by sulfa drugs in the active solutions. The frequency value of the symmetric stretching mode of the sulfonyl group upsilons (SO2) is chosen as a suitable electronic index and measured for the individual active forms in aqueous and Me2SO solutions. The linear correlation that exists between bacteriostatic parameter and vibration frequency (over the complete range of data at present available) proves a strict relationship between electronic structure and bacteriostatic activity in this class of drugs. Furthermore, it justifies the assumption used for the calculation of the bacteriostatic activity of the anionic form; i.e., in equilibrium with a very active species (the anion) a less active species (the neutral form) gives a negligible contribution or does not contribute at all to the total activity. The results can be summarized as follows: the lower the frequency of the symmetric stretching mode of the SO2 group of any active species of sulfonamide type compounds, the higher its bacteriostatic activity. The existence of a clear structure-activity correlation demonstrates that the whole class of compounds, whatever their form, has a single mechanism of action, while incontrovertible deviations from the general trend indicate differences or complications in the mechanism itself, but does not demonstrate that the group on which the structural index is localized plays a dominant role in the biological process. The usefulness of pKa and NH2 proton chemical shift of precursor amine as indirect indices of the electronic structure of the anionic forms is explored on extensive sets of available data.

Redox properties and acid-base equilibria of zucchini mavicyanin.

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.

Axial ligation and polypeptide matrix effects on the reduction potential of heme proteins probed on their cyanide adducts.

The enthalpic and entropic changes accompanying the reduction reaction of the six-coordinate cyanide adducts of cytochrome c, microperoxidase-11 and a few plant peroxidases were measured electrochemically. Once the compensating changes in reduction enthalpy and entropy due to solvent reorganization effects are factorized out, it is found that cyanide binding stabilizes enthalpically the ferriheme following the order: cyochrome c > peroxidase > microperoxidase-11. The effect is inversely correlated to the solvent accessibility of the heme. Comparison of the reduction thermodynamics for the cyanide adducts of cytochrome c and plant peroxidases with those for microperoxidase-11 and myoglobin, respectively, yielded an estimate of the consequences of protein encapsulation and of the anionic character of the proximal histidine on the reduction potential of the heme-cyanide group. Insertion of the heme-CN group into the folded peptide chain of cyt c induces an enthalpy-based decrease in E degrees ' of approximately 100 mV, consistent with the lower net charge of the oxidized as compared to the reduced iron center, whereas a full imidazolate character of the proximal histidine stabilizes enthalpically the ferriheme by approximately 400 mV. The latter value should be best considered as an upper limit since it also includes some solvation effects arising from the nature of the protein systems being compared.

Anion binding to cytochrome c2: implications on protein-Ion interactions in class I cytochromes c.

The binding of several inorganic and carboxylate anions to cytochrome c2 from Rhodopseudomonas palustris has been investigated by monitoring the salt-induced changes in the redox potential of the heme, using an interpretative model based on the extended Debye-Hückel equation. Most anions were found to interact specifically with the protein at one or multiple sites. Binding constants to the oxidized protein in the range 10(1)-10(2) m-1 were determined from the anion concentration dependence of the chemical shift of the isotropically shifted heme methyl resonances. For several anions the stoichiometry and strength of the binding to cytochrome c2 were found comparable with those determined for mitochondrial cytochromes c, in spite of the limited sequence similarity (less than 40%) and the lower positive charge of the bacterial protein. These analogies were interpreted as indicative of the existence of common binding sites which are proposed to be located in the conserved lysine-rich domain around the solvent-exposed heme edge, which is also the surface area likely involved in the interaction with redox partners. The changes in E degrees due to partial neutralization of the positive charge of cytochrome c2 due to specific anion binding were found comparable with those for the mitochondrial species.

Metal-induced conformational heterogeneity of transferrins: a spectroscopic study of indium(III) and other metal(III)-substituted transferrins.

The conformation in solution of three different metal(III)-transferrins, namely aluminum(III), gallium(III) and indium(III) transferrin, was investigated by absorption, CD, 1H NMR and 13C NMR spectroscopies. The formation of the respective metal-transferrin complexes and the characteristic 2:1 metal-to-protein binding stoichiometry were unambiguously demonstrated, in all cases, through UV difference studies. The 13C NMR spectra of these metallotransferrins in the carbonyl region are very similar to one another pointing out that the arrangement of the synergistic anion in the binding site must be essentially the same. However, the CD spectra in the near UV (aromatic region) reveal the occurrence of significant differences between indium transferrin, on one side, and the other two derivatives, on the other. Also, the 1H NMR spectra exhibit a number of different features suggesting the occurrence of metal-induced conformational heterogeneity around the metal sites. Such metal-induced conformational heterogeneity probably affects the transferrin-receptor recognition process, resulting in a different metabolic fate of these metals in the organisms.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in southern Italy: a case of G6PD A(-) associated with favism.

During a routine screening for G6PD deficiency in the Province of Matera (Southern Italy), an eleven-year-old boy was brought to our attention who had fever obviously caused by a viral infection, but who also had hepatosplenomegaly and haemoglobinuria. The boy had previously experienced two severe haemolytic attacks. At the age of six months severe haemolysis occurred after the ingestion of cooked fava beans. At the age of seven years, the haemolytic episode was very likely triggered by oral administration of co-trimoxazole. The G6PD activity level in erythrocyte lysate was clearly defective (25% of normal). The electrophoretic mobility of G6PD was 110% of normal. These data together with those obtained from biochemical and molecular characterisation allowed the variant to be identified as G6PD A(-). This is the first report of an association between the African type G6PD deficiency variant and favism.

Solvent-based deuterium isotope effects on the redox thermodynamics of cytochrome c.

The reduction thermodynamics of cytochrome c (cytc), determined electrochemically, are found to be sensitive to solvent H/D isotope effects. Reduction of cytochrome c is enthalpically more favored in D(2)O with respect to H(2)O, but is disfavored on entropic grounds. This is consistent with a reduction-induced strengthening of the H-bonding network within the hydration sphere of the protein. No significant changes in E degrees ' occur, since the above variations are compensative. As a main result, this work shows that the oxidation-state-dependent differences in protein solvation, including electrostatics and solvent reorganization effects, play an important role in determining the individual enthalpy and entropy changes of the reduction process. It is conceivable that this is a common thermodynamic feature of all electron transport metalloproteins. The isotope effects turn out to be sensitive to buffer anions which specifically bind to cytc. Evidence is gained that the solvation thermodynamics of both redox forms of cytc are sensibly affected by strongly hydrated anions.

Evidence for a conserved binding motif of the dinuclear metal site in mammalian and plant purple acid phosphatases: 1H NMR studies of the di-iron derivative of the Fe(III)Zn(II) enzyme from kidney bean.

The di-iron core of mammalian purple acid phosphatases has been reproduced in the plant enzyme from kidney bean (Mr 111000) upon insertion of an Fe(II) ion in place of the native zinc(II) in the dinuclear Fe(III)Zn(II) core. The shortening of the electronic relaxation time of the metal centre allows detection of hyperfine-shifted 1H NMR resonances, although severe broadening due to Curie relaxation prevents independent signal assignment. Nevertheless, comparison of the spectral features of the structurally characterized plant enzyme with those of the mammalian species, which were previously extensively assigned, is consistent with a close similarity of the metal-binding sites, also suggested by previous sequence-alignment studies. Some differences appear to be mainly localized at the M(II) site. Spectral comparison was also carried out on the Fe(III)Co(II) derivatives.

Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c.

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

Partial purification and characterization of the messenger RNA for human glucose-6-phosphate dehydrogenase.

Glucose-6-phosphate dehydrogenase (G6PD) is a household enzyme that accounts, in many cells, for about 0.03% of cellular protein. We have developed an assay for G6PD-specific mRNA based on in vitro translation of RNA from human fibroblasts and immunoprecipitation of the translation products with an anti-G6PD antiserum. By making use of this assay, G6PD mRNA has been purified 50 to 100-fold in three steps. We estimate that the mRNA encoding G6PD constitutes less than 0.02% of total poly(A)+ RNA in human fibroblasts. The size of the G6PD mRNA has been established in denaturing conditions as being in the range between 2800 and 3200 nucleotides. This has been confirmed by Northern blot analysis. Since the G6PD coding sequence is estimated to be about 1491 nucleotides, the G6PD mRNA has long untranslated sequences, most of which is at the 3' end and which may be heterogeneous in length. The sequence of the last 608 nucleotides of this mRNA has been determined.

Activity of adenosine deaminase allelic forms in intact erythrocytes and in lymphocytes.

The distribution of ADA activity has been studied for ADA 1 and ADA 2-1 individuals in intact and lysed erythrocytes and in lymphocyte lysates. ADA activity in intact RBC is 20-30% lower than in lysates, in lymphocytes it is 10 times higher. The difference between ADA 1 and ADA 2-1 mean enzyme activities which we previously described in RBC lysates has been found also in intact RBC and in lymphocyte lysates.

Nonrandom patterns of codon usage and of nucleotide substitutions in human alpha- and beta-globin genes: an evolutionary strategy reducing the rate of mutations with drastic effects?

Nucleotide substitutions within a structural gene can cause two principal "drastic" phenotypic effects at the protein level: translatable leads to untranslatable and nonpolar hydrophobic in equilibrium hydrophilic amino acid substitutions. The sequence of nucleotides in the structural human alpha- and beta-globin genes and their variants were examined to determine whether codon usage, patterns of nucleotide substitutions, or both, reduced the relative and absolute rates of these unfavorable mutations. Based on translation of abnormal hemoglobins, it is likely that all 61 nontermination codons are potentially translatable, though only 47 are normally used. Moreover, codons that can mutate to a termination codon are never used whenever the corresponding amino acid is specified also by triplets that cannot mutate to termination by a single-step mutation. Thus, the number of opportunities to mutate to an untranslatable codon is reduced to the minimum compatible with the amino acid composition of these chains. The relative rates of U in equilibrium non-U substitutions were much lower than those of other substitutions. Because U residues must be involved in most termination mutations and in all nonpolar hydrophobic in equilibrium hydrophilic amino acid substitutions, there is a considerable reduction of mutational events, causing drastic phenotypic effects. These findings are likely to be the end result of evolutionary selection by yet unknown mechanisms.

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c.

The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50 degrees C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E degrees' observed with increasing ionic strength [DeltaE degrees'IS = (E degrees')I = 0.1 M - (E degrees')I = (0)M = -0.035 V at 25 degrees C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factored out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK(a) for a residue deprotonation is the key event of this conformational change.

Effects of specific anion-protein binding on the alkaline transition of cytochrome c.

The thermodynamic parameters of the alkaline transition of beef heart ferricytochrome c have been measured through direct electrochemistry experiments carried out at variable pH and temperature in the presence of different sulfate concentrations. Sulfate is known to bind specifically to cytochrome c in a sequential manner at two surface sites. The effects of such a specific binding reflect on the thermodynamics of the transition and can be satisfactorily interpreted within the frame of the Debye-Hückel theory with simple electrostatic considerations. In particular, the increase in the thermodynamic pKa values (extrapolated to I = 0) upon sulfate binding turns out to be a fully enthalpic effect which can be accounted for by considering the coulombic effects of the formation of ionic couple(s) on the protein surface. This study also shows that the apparent pKa values at finite ionic strength are only moderately affected by the nature of the anion in solution, and differences tend to vanish at high ionic strength.

Isolation and characterization of two peroxidases from Cucumis sativus.

Two heme peroxidases of 35.2 and 36.5 kDa have been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and 1H NMR spectra in the pH range 3.5-10.5. Their spectroscopic and catalytic properties, which are closely similar, are characteristic of highly homologous isoenzymes. Both proteins, as isolated, exist as a mixture of two ferric forms containing a high-spin and a low-spin heme in an approximately 2:1 molar ratio. The latter form likely contains a hydroxide ion axially coordinated to the heme iron and is proposed to be the result of partial irreversible protein inactivation due to the purification procedure. Both proteins in the reduced form are fully high-spin. The high-spin ferric form is sensitive to two acid-base equilibria with apparent pKa values of approximately 5 and 8.5, which have been assigned to the distal histidine and the arginine adjacent to it, respectively. These equilibria also affect the catalytic activity and the interaction with inorganic anions such as azide and fluoride. The reactivity of both proteins is closely similar to that of other plant peroxidases, primarily horseradish peroxidase; however, they also show spectroscopic properties similar to those of cytosolic ascorbate peroxidase. Therefore, overall, these two species show molecular, spectroscopic and catalytic features which are rather peculiar among plant peroxidases.

Magnetic resonance of Fe-S clusters: isolation and characterization of a 7Fe ferredoxin from Rhodopseudomonas palustris.

A novel iron-sulfur protein from the photosynthetic purple bacterium Rhodopseudomonas palustris was purified to homogeneity and identified as a ferredoxin on the basis of its physicochemical properties. Based on the uv/vis spectrum, iron quantitation, cyclic voltammetry, EPR, and 1H NMR data, the ferredoxin is found to contain two iron-sulfur clusters, one [3Fe-4S] and one [4Fe-4S], which places this protein in the class of 7Fe ferredoxins. The voltammetric peak potentials of the two clusters are -0.260 and -0.560 V at pH 8.0. The molecular mass around 19 kDa makes this protein the heaviest known in this class. This paper further demonstrates the diagnostic power of magnetic resonance spectroscopies in recognition of the two types of clusters in iron-sulfur proteins.

Influence of surface charges on redox properties in high potential iron-sulfur proteins.

The pH-dependence of the reduction potential determined through differential pulse voltammetry for the high potential iron sulfur proteins (HiPIP) from R. globiformis, C. vinosum, R. gelatinosus, E. vacuolata (I and II), E. halophila (I and II) is reported. A decrease in reduction potential with pH is invariably observed in the pH range where deprotonation of the imidazolium nitrogen of histidine residue(s) occurs. No pH dependence is observed for the only protein lacking histidines. It appears that surface charges like the His imidazolium groups are capable of influencing the reduction potential despite the known quencing of the electrostatic interactions due to solvent effects.

Cyclic voltammetry and 1H-NMR of Rhodopseudomonas palustris cytochrome c2 pH-dependent conformational states.

The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c2 from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5-11, the E degree values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine-shifted 1H-NMR resonances of the protein are sensitive to a number of acid/base equilibria. In particular, four equilibria have been determined for the oxidized protein with pKa values of 2.5, 5.5, 6.6 and 9.0. The lowest pKa most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pKa is associated with a low-pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pKa of 6.6 modulates the E degree values without determining any detectable spectral change and most likely involves the acid/base equilibrium of an histidine residue in close vicinity of the heme (possibly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (most probably N-donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic constant of 0.98 s-1 at pH 9.2.