The nucleotide-independent Fe(III)-binding site is located on beta subunit of the mitochondrial F(1)-ATPase.

Upon separation of a crude preparation of beta subunit ("beta fraction") from mitochondrial F(1)-ATPase containing one equivalent of Fe(III) in the nucleotide-independent site (1Fe(III)-loaded MF(1)), Fe(III) is almost completely recovered. CD spectra show that "beta fraction" maintains the structural changes induced by Fe(III) in the whole enzyme. In accordance, EPR reveals that the Fe(III) site geometry is conserved in "beta fraction." Moreover, the EPR spectra of 1Fe(III)-loaded MF(1) and its "beta fraction" undergo similar changes of the line-shape upon Pi binding at the catalytic site, indicating that the Pi and Fe(III) are proximal on beta. Highly purified beta in nucleotide-free form binds 1mol of Fe(III)/mol of protein. MF(1) "freezed" by inhibitors with two beta in closed conformation and one beta in open or half-closed conformation binds 1mol of Fe(III)/mol of enzyme. Therefore, the Fe(III) site location in the unique beta subunit not adopting the closed conformation is proposed.

Human glutathione transferase P1-1 and nitric oxide carriers; a new role for an old enzyme.

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i) < 10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.

Anion size modulates the structure of the A state of cytochrome c.

Several studies have shown that anions induce collapse of acid-denatured cytochrome c into the compact A state having the properties of the molten globule and that the anion charge is the main determinant for the A state stabilization. The results here reported show that the anion size plays a role in determining the overall structure of the A state. In particular, small anions induce formation of an A state in which the native Met80-Fe(III) axial bond is recovered and the nativelike redox properties restored. On the other hand, the A state stabilized by large anions shows a histidine (His26 or His33) as the sixth ligand of the heme-iron, a very weak interaction between Trp59 and the heme propionate, and lacks nativelike redox properties. The two anion-stabilized states show similar stability, indicating that (i) the hydrophobic core (which is equally stabilized by all the anions investigated, independently of their size) is the region that mainly contributes to the macromolecule stabilization, and (ii) the flexible loops are responsible for the spectroscopic (and, thus, structural) and redox differences observed.

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene.

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.

The heme-containing N-fragment (residues 1-56) of cytochrome c is a bis-histidine functional system.

The structural and redox properties of a heme-containing fragment (1-56 residues) of cytochrome c have been investigated by spectroscopic (circular dichroism, electronic absorption, and EPR) and voltammetric techniques. The results indicate that the N-fragment lacks ordered secondary structure and has two histidines axially bound to the heme-iron (the native His18 and a misligated His26 or His33). Despite the absence of ordered secondary structure, the peptide chain shields the heme group from solvent, as shown by (i) the pK(a) of protonation of the nonnative histidine ligand (5.18 +/- 0.05), lower than that of the bis-histidine guanidine-unfolded cytochrome c (5.58 +/- 0.05), and (ii) the redox potential, E(o) = 0 +/- 5 mV versus NHE, close to that of bis-histidine cytochrome c mutants but less negative than that of bis-histidine complexes of microperoxidase with short peptides. The electroactive N-fragment may be taken as a "minichrome c" model, with interesting potential for application to biosensor technology; further, the system provides useful information for a deeper understanding of cytochrome c folding and structural/functional organization.

EPR detection of protein-derived radicals in the reaction of H(2)O(2) with Fe bound in mitochondrial F(1)ATPase.

A severe inactivation is obtained upon the addition of H(2)O(2) to bovine heart F(1)ATPase samples containing Fe(III) in the nucleotide-independent site, and Fe(II) in the ATP-dependent site. EPR spectra at 4.9 K of these samples indicate that H(2)O(2) produces the complete oxidation of Fe(II) to Fe(III) and the concomitant appearance of two protein-derived radical species. The two signals (g = 2.036 and g = 2.007) display a different temperature dependence and saturation behavior. The relaxation properties of the radical at g = 2.036 suggest magnetic interaction with one of the two iron centers. Such events are not observed when H(2)O(2) is added either to native F(1)ATPase containing a high amount of Fe(II) and low amount of Fe(III) or to F(1)ATPase deprived of endogenous Fe and subsequently loaded with only Fe(III) in both sites. It is hypothesized that in F(1)ATPase samples containing both Fe(III) and Fe(II), intramolecular long-range electron transfer may occur from Fe(II) to a high oxidation state species of Fe formed in the nucleotide-independent site upon oxidation of Fe(III) by H(2)O(2).

Formation of a molten-globule-like state of cytochrome c induced by high concentrations of glycerol.

The effect of glycerol on the structure of cytochrome c was investigated by circular dichroism, absorbance and EPR spectroscopy. The results obtained show that an increasing concentration of the organic solvent (70-99.2%, v/v) in aqueous-polyalcohol mixtures converts native cytochrome c into a new, low spin form through a fully reversible, two-state transition. The glycerol-stabilized form (that we call here the G state) retains native-like amounts of alpha-helix structure while rigid tertiary structure and native Fe(III)-Met(80) axial bond are lost. Analysis of data suggests a molten globule character of the G state; support to this view is afforded by the striking similarities between the spectroscopic (and, thus, structural) properties of the G state with those of the acidic molten globule of the protein (A state).

Functional modulation of the Thr72-->Ile mutant from Scapharca inaequivalvis homodimeric hemoglobin.

The pH and temperature dependence of both the kinetic and thermodynamic properties of the Thr72-->Ile mutant of Scapharca inaequivalvis homodimeric hemoglobin were investigated between pH 2 and 10 and between 8 degrees C and 36 degrees C, in comparison with the wild-type recombinant protein. Results demonstrate pH-independent O2-binding properties, at least between pH 5 and 10, with the higher affinity of the mutant being related to a less negative entropy change. This observation may relate to a variation in the number of water molecules involved in the intersubunit communication. Furthermore, the kinetic properties of ligand association and dissociation seem to be in keeping with possible structural alterations of water molecules at the subunit interface occurring in the Thr72-->Ile mutant as well as with amino acid residues involved in the modulation of reactivity and cooperativity at the level of (1) the proximal side of the heme pocket and of (2) the heme propionates bridging the two subunits.

Cu,Zn superoxide dismutase from Photobacterium leiognathi is an hyperefficient enzyme.

The catalytic rate constant of recombinant Photobacterium leiognathi Cu,Zn superoxide dismutase has been determined as a function of pH by pulse radiolysis. At pH 7 and low ionic strength (I = 0.02 M) the catalytic rate constant is 8.5 x 10(9) M-1 s-1, more than two times the value found for all the native eukaryotic Cu,Zn superoxide dismutases investigated to date. Similarly, Brownian dynamics simulations indicate an enzyme-substrate association rate more than two times higher than that found for bovine Cu,Zn superoxide dismutase. Titration of the paramagnetic contribution to the water proton relaxation rate of the P. leiognathi with increasing concentration of halide ions with different radii indicates that the proteic channel delimiting the active site is wider than 4.4 A. This is at variance with that found on the eukariotic enzymes, and provides a rationale for the high catalytic rate of the bacterial enzyme. Evidence for solvent exposure of the active site different from that observed in the eukaryotic enzyme is suggested from the pH dependence of the water proton relaxation rate and of the EPR spectrum line shape, which indicate the occurrence of a prototropic equilibrium at pH 9.1 and 9.0, respectively. The pH dependence of the P. leiognathi catalytic rate has a trend different from that observed in the bovine enzyme, indicating that groups differently exposed to the solvent are involved in the modulation of the enzyme-substrate encounter.

Cooperative mechanism in the homodimeric myoglobin from Nassa mutabilis.

Oxygen binding and spectroscopic properties of the homodimeric myoglobin (Mb) from the prosobranchia sea snail Nassa mutabilis have been investigated. Oxygen equilibrium curves are pH-independent and cooperative with P50 = 5 +/- 1 mmHg and n approximately 1.5. Circular dichroism spectra of the oxygenated and deoxygenated form of N. mutabilis Mb are superimposable between 190 and 250 nm, suggesting a mechanism for cooperative ligand binding that does not involve changes in the alpha-helical content of the whole protein. The oxygen dissociation process is biphasic and pH-dependent, with different pKa values (=6.7 +/- 0.2 and 8.5 +/- 0.3) for the two phases. Moreover, the activation energy is essentially the same for both oxygen dissociation processes (Ea = 56.4 +/- 2.1 kJ/mol for the fast phase, and Ea = 53.8 +/- 1.9 kJ/mol for the slow phase), indicating that the rate difference for O2 dissociation between the diliganded and the monoliganded species is mostly dependent on a variation of the activation entropy. Ferrous nitrosylated N. mutabilis Mb shows, at alkaline and neutral pH, axial and rhombic X-band EPR signals, respectively, which display below pH 6 a three-hyperfine pattern typical of five-coordination. The results presented here suggest that in N.mutabilis Mb the kinetic control of cooperativity operates through a mechanism never observed before in other hemoproteins, which requires a ligand-linked large enhancement for the value of the oxygen association process in a molecule not undergoing changes in quaternary structure.

Role of the dimeric structure in Cu,Zn superoxide dismutase. pH-dependent, reversible denaturation of the monomeric enzyme from Escherichia coli.

To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated. Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein. Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface. The melting temperature of the E. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66 degrees C at pH 7.8 and at 79.3 degrees C at pH 6.0. The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources. Apo-superoxide dismutase from E. coli was shown to be nearly as stable as the bovine apoenzyme, whose holo form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes. Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E. coli enzyme was shown to be reversibly perturbed by urea. These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the beta-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E. coli enzyme are due to higher flexibility and accessibility to the solvent of its active-site region.

Spectroscopic characterization of recombinant Cu,Zn superoxide dismutase from Photobacterium leiognathi expressed in Escherichia coli: evidence for a novel catalytic copper binding site.

Cu,Zn superoxide dismutase from Photobacterium leiognathi has been cloned and expressed in Escherichia coli. The circular dichroism spectrum in the UV region of the recombinant protein indicates an higher content of random coil structure with respect to the eukaryotic enzymes. Investigation of the active site by optical, CD, and EPR spectroscopy indicates a different coordination geometry around the catalytic copper site with respect to the eukaryotic enzymes. In particular a different orientation of the metal bridging histidine is suggested. The pH dependence of the copper EPR spectrum shows the presence of a single equilibrium which is at least one unit lower than the pK value observed for the bovine enzyme. Despite such structural differences the catalytic rate of this enzyme is identical to that observed for the eukaryotic Cu,Zn superoxide dismutase, suggesting that the overall electric field distribution is similar to that observed in the eukaryotic enzymes.

Solvent effects on the distribution of conformational substates in native and azide reacted Cu, Zn superoxide dismutase. An EPR study.

Native and azide reacted Cu, Zn superoxide dismutase in aqueous and mixed water-glycerol solution have been investigated by EPR spectroscopy at low temperature. An accurate computer simulation, based on a well established theoretical model which has been reformulated for rhombic symmetry, has shown that the EPR spectrum of the copper ion in the native protein shows a significant g and A strain in the parallel region. The strain arises from a distribution of the ligand field strengths onto the metal ion and this could be traced back to the existence of a multiplicity of conformational states in the protein molecule. The strain is reduced in the presence of azide which is known to bind directly to the copper atom and to give rise to a more relaxed configuration corresponding to a square pyramidal geometry in which the apical ligand occupies an elongated position. In both samples, addition of glycerol further reduces the strain, indicating that the solvent is directly coupled to the protein matrix, thereby modulating the structural heterogeneity displayed by the protein molecule.

Characterization of the binding of Fe(III) to F1ATPase from bovine heart mitochondria.

The binding Fe(III) to F1ATPase purified from beef heart mitochondria has been characterized by chemical analyses and EPR spectroscopy. F1ATPase binds 2 mol of Fe(III)/mol of protein selectively in the presence of saturating concentrations of ATP. In the absence of nucleotides or in the presence of either saturating ADP or limiting ATP concentrations, the enzyme binds 1 equivalent of Fe(III). F1ATPase pretreated with 5'-p- fluorosulfonylbenzoyladenosine, that selectively modifies the non-catalytic sites, binds only 1 mol of Fe(III)/mol of protein in the presence of either saturating ATP or ADP, Fe(III)-loaded F1ATPase containing either 1 or 2 equivalents of Fe(III) show identical EPR signals at g=4.3. The signals are not perturbed by the binding of nucleotides to the enzyme while they are altered by phosphate addition. These results indicate that F1ATPase contains two distinct Fe(III)-binding sites, which differ from nucleotide-binding sites, and that one of these sites is opened up for Fe(III) uptake by conformational changes induced by binding of ATP to the loose non-catalytic site.

Metal uptake of recombinant cambialistic superoxide dismutase from Propionibacterium shermanii is affected by growth conditions of host Escherichia coli cells.

We constructed the complete nucleotide sequence coding for the cambialistic superoxide dismutase from Propionibacterium shermanii by ligation of a synthetic linker to a polymerase chain reaction amplification product obtained using degenerate primers. We set up an expression system yielding large amounts of recombinant superoxide dismutase in the cytoplasm of Escherichia coli and purified the enzyme from cells grown in a complex medium. The physicochemical properties of the recombinant enzyme were identical to those of the natural protein. Under anaerobic conditions the enzyme produced in an iron-supplemented medium incorporated iron as metal cofactor, while the enzyme purified from cells grown under aerobic conditions contained a variable amount of iron and manganese depending on metal availability. Functional equivalence of the two metals in this superoxide dismutase variant was indicated by independence of enzyme activity from Fe/Mn ratio.

A single mutation (Thr72-->Ile) at the subunit interface is crucial for the functional properties of the homodimeric co-operative haemoglobin from Scapharca inaequivalvis.

The in vivo expression and the functional and spectroscopic properties are reported for a mutant of the homodimeric haemoglobin of the mollusc Scapharca inaequivalvis (HbI), where residue threonine 72 (position 9 in the E helix) at the subunit interface has been substituted by isoleucine. The aim of this study is to test the hypothesis that increasing the hydrophobicity character of the subunit interface may modulate oxygen affinity and co-operativity of this haemoglobin. In fact, X-ray crystal structure studies have shown that the subunit interface, formed by the E and F helices of the two chains, changes its character from hydrophilic to hydrophobic upon oxygenation. This is primarily due to extrusion of Phe97 side-chain from the haem pocket toward the interface, which disrupts a network of ordered water molecules and results in close van der Waals contacts between Phe97 and Thr72 of the partner subunit. Thr72-->Ile HbI was expressed in E. coli after mutation of HbI-DNA and it displays a approximately 40-fold enhancement of oxygen affinity and a marked reduction of co-operativity in oxygen binding, with respect to native HbI. These functional properties and the kinetics of oxygen dissociation and carbon monoxide combination rates, as well as data from EPR and circular dichroism spectroscopy, indicate that indeed the increase of the hydrophobicity at the interface upon mutation stabilizes the "high affinity" conformation of the protein, suggesting that extrusion of Phe97 toward the interface should be facilitated even in the unliganded form.

Impaired copper binding by the H46R mutant of human Cu,Zn superoxide dismutase, involved in amyotrophic lateral sclerosis.

Several point mutations in the gene coding for human Cu,Zn superoxide dismutase have been reported as being responsible for familial amyotrophic lateral sclerosis (FALS). However, no direct demonstration has been provided for a correlation between total superoxide dismutase activity and severity of the FALS pathology. In order to get a better insight into the mechanism(s) underlying the FALS phenotype, we have investigated the activity and the copper binding properties of the single mutant H46R, which is associated with a Japanese form of FALS. We have shown that this mutant is structurally stable but lacks significant enzyme activity and has impaired capability of binding catalytic copper. The mutant protein can be fully reconstituted with copper in vitro but its ESR spectrum displays an axial shape quite different from that of the wild-type.

Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative: a spectroscopic and thermodynamic study.

Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated quantitatively by EPR and absorbance spectroscopy. The X-band EPR spectra and the absorption spectra in the Soret region of the nitrosylated derivative of ferrous native and carboxymethylated cytochrome c display the same basic characteristics reported for the beef heart cytochrome a3 in cytochrome c oxidase, and horseradish and baker's yeast cytochrome c peroxidase, as well as the high affinity form of oxygen carrying proteins. Values of the dissociation equilibrium constant for nitrosylation of ferrous native and carboxymethylated cytochrome c are 8.2 x 10(-6) M and < or = 5 x 10(-8) M, respectively, at pH 7.0 and 10 degrees C. The results here reported represent clearcut evidence for the nitric oxide-induced cleavage of the Fe-Met80 bond in ferrous native cytochrome c, and allow estimation of the free energy associated to the heme-iron sixth coordination bond (> 10 kJ mol-1, at 10 degrees C).

Mini-myoglobin: native-like folding of the NO-derivative.

Mini-myoglobin is a polypeptide fragment (residues 32-139) obtained by limited proteolysis of horse heart apomyoglobin and reconstituted with the natural heme. Its functional properties are very similar to those of native myoglobin and therefore it may represent a model for testing the functional role of the protein fragment encoded by the central exon of myoglobin gene (residues 31-105). Here we have investigated some properties of the nitric oxide derivative of mini-myoglobin in comparison with those of NO-myoglobin, to provide more structural information on the heme pocket residues in addition to that already acquired by electron paramagnetic resonance of the cobalt-substituted mini-myoglobin. At pH 7.0, optical and circular dichroism Soret spectra, as well as electron paramagnetic resonance spectra reveal that the heme orientation in the pocket and the coordination state of the ferrous iron in NO-mini-myoglobin are similar to those of the whole protein. The spectra of the NO-mini-myoglobin complex are very sensitive to pH changes at variance to what is observed for the NO-myoglobin derivative in the same pH range (5.5-9.5). In particular, increasing or decreasing pH from 7.0, results in a decrease of the extinction coefficient and of the ellipticity in the Soret region and in a change of the shape of the electron paramagnetic resonance signal. The spectral changes are diagnostic for a transition from a hexa-coordinated (at pH 7.0) to a penta-coordinated heme (at pH 5.5 or 9.5), with the proximal histidine-iron bond either broken or stretched dramatically. Thus, although mini-myoglobin is able to bind NO in a geometry similar to that of the native protein, the resulting NO derivative shows a much higher pH dependence, suggesting that the two lacking side domains are mainly involved in enhancing the stability of the hemoprotein core.

Functional, spectroscopic and structural properties of haemoglobin from chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex).

The functional and spectroscopic properties of chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex) haemoglobin (Hb) have been studied with special reference to the action of allosteric effectors and temperature. Moreover, the amino acid sequences of the N-terminal segments of the alpha- and beta-chains have been determined. The present results indicate that chamois and steinbock Hbs display a low affinity for O2, which appears to be modulated in vivo by Cl- ions rather than 2,3-bisphosphoglycerate. The Bohr effect for O2 binding to chamois and steinbock Hb is higher than for reindeer and bovine Hbs, being similar to that of human Hb. Moreover, the temperature-dependence of oxygenation appears intermediate between that of human and reindeer Hbs. E.p.r. and absorption spectroscopic properties of the ferrous nitrosylated derivative of chamois and steinbock Hbs suggest that both haemoproteins are in a low-affinity conformation even in the absence of InsP6. The reduced effect of polyphosphates on the functional and spectroscopic properties of chamois and steinbock Hb agree with amino acid differences in the N-terminal segment of the beta-chains (i.e. the deletion of Val(NA1) and the replacement of His(NA2), present in human Hb, and Gln(NA2), present in horse Hb, by Met). The molecular mechanism modulating the basic reaction of O2 with chamois and steinbock Hb may be linked to specific physiological needs related to the high-altitude habitats of these two animals.