Structural studies of mitochondrial coupling factor 1 using tyrosine fluorescence

The absorbance and fluorescence spectral properties of mitochondrial F1-ATPase confirm that this protein does not contain tryptophan residues and therefore its fluorescence is due to tyrosines. The 36% increase in the fluorescence and the almost 100% increase in quantum yield upon denaturation of the protein suggest that a considerable number of tyrosyl residues have a very low quantum yield in the native enzyme. Quenching experiments using iodide indicate that all of the fluorophores are quenched and also all of them with the same quenching constant. These observations are interpreted as confirmatory of what has been found with several other proteins whose fluorescence originates from tyrosyl residues, where the buried tyrosines fluoresce with a much lower quantum yield than those which are exposed. ATP added to F1 previously depleted of loosely bound nucleotides changes the quenching constant of iodide and the quantum yield and this is interpreted to be due to a conformational change induced by the binding of the nucleotide to the enzyme. Addition of 2-mercaptoethanol decreases, although slightly, the polarization of the fluorescence. However, SDS addition gives a much bigger decrease. Hence disulphide bridges are less important for the tertiary structure of the protein than hydrophobic interactions, hydrogen bonding or other forces. Nevertheless the conformational change induced by reduction of disulphide bridges is detected in iodide quenching experiments and the change of the quantum yield of the enzyme.     

Spectroscopic studies on the tyrosine residues of canavalin.

Canavalin is a tetramer with 6 tyrosines per subunit. In the work presented here, we have classified these tyrosines by their spectrophotometric and fluorometric pH titration and their ability to be quenched I-. Of the 6 residues, 2 were found to be exposed to the solvent. One (pK = 10.2) contributes 28% of the total fluorescence intensity; the second has a pK of 11.50, and a lower quantum yield, contributing only 16% of the total intensity. The remaining 4 residues (pK = 12.5, contributing 54% of fluorescence intensity) are buried; their titration is irreversible, requiring protein denaturation.     

Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization.

The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the tryptophan residue in apoazurin has the same asymmetric environment both in the ground and excited states. This finding suggests tight packing of the protein at the tryptophan environment. Native azurin does not decay monoexponentially. Moreover, comparison between the quantum yield calculated from the decay kinetics and the one measured directly shows that the majority of the azurin molecules are not fluorescent. There is thus variability in the structure of azurin molecules with an equilibration time that is longer than the fluorescence lifetime. Different asymmetric environment was found for the tryptophan residue in oxidized and reduced holoprotein and in apoazurin, as studied by the circular polarization of the fluorescence. D(2)O increases the fluorescence lifetime of apoazurin by 6 percent, compared to the lifetime in H(2)O solution; therefore water molecules may have access to the tryptophan residue, though the latter is situated in a hydrophobic environment.     

Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy.

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.     

Studies of Pseudomonas aeruginosa azurin mutants: cavities in beta-barrel do not affect refolding speed

Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold. Removal of copper produces an apoprotein with the same structure as holoazurin. To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a beta-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins. The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins. The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect. Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin. In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin. We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.     

Dynamic fluorescence in copper proteins Selected examples

Summary The fluorescence properties of three copper proteins, namely human superoxide dismutase,Pseudomonas aeruginosa azurin andThiobacillus versutus amicyanin have been studied. All these proteins show a non-exponential decay of fluorescence, though the tryptophanyl residues responsible for the emission are very differently located in the three proteins. All the three decays can be fitted by at least two lifetimes or better with one or two lorentzian-shaped, continuous distributions of lifetime. In each case the removal of copper affects the quantum yield of fluorescence without affecting the shape of the emission.     

Solution structural studies of the Saccharomyces cerevisiae TATA binding protein (TBP)

The intrinsic fluorescence of the six tyrosines located within the C-terminal domain of the Saccharomyces cerevisiae TATA binding protein (TBP) and the single tryptophan located in the N-terminal domain has been used to separately probe the structural changes associated with each domain upon DNA binding or oligomerization of the protein. The unusually short-wavelength maximum of TBP fluorescence is shown to reflect the unusually high quantum yield of the tyrosine residues in TBP and not to result from unusual tryptophan fluorescence. The anisotropy of the C-terminal tyrosines is very high in monomeric, octameric, and DNA-complexed TBP and comparable to that observed in much larger proteins. The tyrosines have low accessibility to an external fluorescence quencher. The anisotropy of the single tryptophan located within the N-terminal domain of TBP is much lower than that of the tyrosines and is accessible to an external fluorescence quencher. Tyrosine, but not tryptophan, fluorescence is quenched upon TBP-DNA complex formation. Only the tryptophan fluorescence is shifted to longer wavelengths in the protein-DNA complex. In addition, the accessibility of the tryptophan residue to the external quencher and the internal motion of the tryptophan residue increase upon DNA binding by TBP. These results show the following: (i) The structure of the C-terminal domain structure is unchanged upon TBP oligomerization, in contrast to the N-terminal domain [Daugherty, M. A., Brenowitz, M., and Fried, M. G. (2000) Biochemistry 39, 4869-4880]. (ii) The environment of the tyrosine residues within the C-terminal domain of TBP is structurally rigid and unaffected by oligomerization or DNA binding. (iii) The C-terminal domain of TBP is uniformly in close proximity to bound DNA. (iv) While the N-terminal domain unfolds upon DNA binding by TBP, its increased correlation time shows that the overall structure of the protein is more rigid when complexed to DNA. A model that reconciles these results is proposed.     

Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence.

Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.     

Conformational heterogeneity of the copper binding site in azurin. A time-resolved fluorescence study.

Comparison of the fluorescence spectra and the effect of temperature on the quantum yields of fluorescence of Azurin (from Pseudomonas fluorescens ATCC-13525-2) and 3-methylindole (in methylcyclohexane solution) provides substantive evidence that the tryptophan residue in azurin is completely inaccessible to solvent molecules. The quantum yields of azurin (CuII), azurin (CuI), and apoazurin (lambda ex = 291 nm) were 0.052, 0.054, and 0.31, respectively. Other evidence indicates that there is no energy transfer from tyrosine to tryptophan in any of these proteins. The fluorescence decay behavior of each of the azurin samples was found to be invariant with emission wavelength. The fluorescences of azurin (CuII) and azurin (CuI) decay with dual exponential kinetics (tau 1 = 4.80 ns, tau 2 = 0.18 ns) while that of apoazurin obeys single exponential decay kinetics (tau = 4.90). The ratio of pre-exponentials of azurin (CuII), alpha 1/alpha 2, is found to be 0.25, and this ratio increases to 0.36 on reduction to azurin (CuI). The results are interpreted as originating from different interactions of the tryptophan with two conformers of the copper-ligand complex in azurin.     

Displacement of the human apoprotein A-I by the human apoprotein A-II from complexes of (apoprotein A-I)-phosphatidylcholine-cholesterol

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24 degrees C. 28 degrees C and 32 degrees C with either pure dimyristoyl-glycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 2, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid-cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II.     

Intrinsic fluorescence of the bacterial copper-containing protein amicyanin

The fluorescence properties of the single tryptophanyl residue present in amicyanin from Thiobacillus versutus are very similar to those of azurin from Pseudomonas aeruginosa and other mononuclear blue copper proteins. The emission maximum is well structured and centered at 318 nm. The quantum yield is strongly affected by the presence of copper, the removal of which is accompanied by a more than sixfold increase in fluorescence, without change in shape. The fluorescence decay of holo-amicyanin is heterogeneous with a longer component of 5.7 ns and a shorter one of 0.7 ns accounting for 90% of the total emitting molecules. Copper-free amicyanin shows instead a single exponential decay (3.3 ns) of intrinsic fluorescence. This lifetime decreases as the temperature increases as does the longer lifetime component of holoamicyanin.     

Properties of Paracoccus denitrificans amicyanin

Paracoccus denitrificans synthesizes an inducible, periplasmic, blue copper protein [Husain, M., & Davidson, V.L. (1985) J. Biol. Chem. 260, 14626-14629] that can be classified as an amicyanin on the basis of its ability to accept electrons from methylamine dehydrogenase. The amino acid composition and sequence of the 10 N-terminal residues of this protein have been determined. From these data, it is evident that amicyanin is structurally distinct from azurins as it contains no disulfide bond and an N-terminal sequence that is completely different from the highly conserved N-terminal azurin sequences. Dialysis of reduced amicyanin against potassium cyanide resulted in a nearly quantitative yield of apoamicyanin. Amicyanin and apoamicyanin exhibit fluorescence emission maxima at 314 nm when excited at 280 nm. Addition of 6 M guanidine hydrochloride shifts these emission maxima to 350 nm. The fluorescence intensity of apoamicyanin is 10-fold greater than that of amicyanin. Addition of copper to the apoprotein caused a stoichiometric quenching of fluorescence and restoration of visible absorbance with no concomitant change in absorbance at 280 nm. At least one cysteine residue, which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) in apoamicyanin, does not react in the holoprotein, even in the presence of 6 M guanidine hydrochloride. Reductive and oxidative titrations of amicyanin indicate that it is a one-electron carrier. This amicyanin is also able to accept electrons from the methylamine dehydrogenase isolated from bacterium W3A1, which is taxonomically very different from P. denitrificans.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins

We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.     

Design, production, and characterization of recombinant neocarzinostatin apoprotein in Escherichia coli

Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.     

Fluorescence studies on the coat protein of alfalfa mosaic virus

The intrinsic luminescence of different forms of the alfalfa mosaic virus (AMV) strain 425 coat protein has been studied, both statically and time resolved. It was found that the emission of the protein (Mr 24,250), which contains two tryptophans at positions 54 and 190 and four tyrosines, is completely dominated by tryptophan fluorescence. The high fluorescence quantum yield indicates that both tryptophans are emitting. Surprisingly, the fluorescence decay is found to be strictly exponential, with a lifetime of 5.1 nsec. Similar results were obtained for various other forms of the protein, i.e. the 30-S polymer, the mildly trypsinized forms of the protein lacking the N-terminal part and the protein assembled into viral particles. Virus particles and proteins of stains S and VRU gave similar results, as well as the VRU protein polymerised into tubular structures. The fluorescence decay is also monoexponential in the presence of various concentrations of the quenching molecules acrylamide and potassium iodide. Stern-Volmer plots were linear and yield for the coat protein dimer with acrylamide a quenching constant of 4.5* 10(8) M-1 sec-1. This indicates that the tryptophans are moderately accessible for acrylamide. For the 30-S polymer a somewhat smaller value was found, whereas in the viral Top a particles the accessibility of the tryptophans is still further reduced. From the decay of the polarisation anisotropy of the fluorescence of the coat protein dimer the rotational correlation time was obtained as 35 nsec. Since this roughly equals the expected rotational correlation time of the dimer as a whole, it suggests that the tryptophans are contained rigidly in the dimer. The results show that in the excited state of the protein the two tryptophans are strongly coupled and suggest that the trp-trp distance is smaller than 10 A. Because the coat protein occurs as a dimer, the coupling can be inter- or intramolecular. The implications for the viral structure are discussed.     

A kinetic study of the reconstitution of azurin from Cu(II) and the apoprotein.

The kinetics of reconstitution of Pseudomonas aeruginosa azurin from its apoprotein and copper(II) salts have been studied using absorbance at 625 nm and fluorescence emission at 308 nm as monitors of the process. At low Cu(II) concentrations the rates of both absorbance and fluorescence changes are linearly dependent on Cu(II) concentration. At higher Cu(II) concentrations the rate of absorbance change is independent of Cu(II) concentration. The rates of both absorbance and fluorescence changes as a function of pH suggest that the titration of a single ionizable group is important for the Cu(II)-dependent reaction. Overall analysis of the kinetics suggests that the fluorescence change and the absorbance change are associated with at least two steps in the overall pathway of the formation of the metal-protein complex, and that the copper(II) and tryptophan environments in this protein, though perhaps spatially close, may be distinct.     

Time-resolved fluorescence study of azurin variants: conformational heterogeneity and tryptophan mobility.

Time-resolved fluorescence and time resolved fluorescence anisotropy studies have been performed on wild-type azurin from Pseudomonas aeruginosa and two variants to study the mobility of Trp48. The two azurin variants in which the microenvironment of Trp48 was changed comprised the single mutations Ile7Ser and Phe110Ser. The experiments were performed on the holo-Cu(I), holo-Cu(II), and apo- forms at various pH values, viscosities, and temperatures; two distinct parts of the emission spectrum were selected for detection. Two prominent subnanosecond lifetimes in the fluorescence decays of the Cu(II) proteins could be observed. The decay of apo-azurin also consists of more than one component. The occurrence of more than one component in the fluorescence decays is explained by conformational heterogeneity. The anisotropy decay results appeared to be different for wild-type and mutated azurins. Phe110Ser and Ile7Ser azurin show more mobility of the Trp48 residue, as reflected in the order parameter.     

Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli

The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. A purification procedure was developed to isolate the azurin obtained from the E. coli cells. No differences were observed between azurins isolated from P. aeruginosa and E. coli. A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01). The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin. Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics). However, it has lost the ability to bind Cu-ions. It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117. In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence decay observed for holo-azurin is not due to the presence of azurin*, but most likely originates from conformational heterogeneity of the holo-azurin.     

The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry–Eyring model, NUF, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 °C, while the unfolding free energy, ΔG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed ΔG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E_a, as derived from the kinetic stability analysis.