Solvent Isotope Effects on Azurin Thermal Unfolding - American

J. Phys. Chem. B 1998, 102, 1021-1028

1021

Solvent Isotope Effects on Azurin Thermal Unfolding R. Guzzi and L. Sportelli* Laboratorio di Biofisica Molecolare, Dipartimento di Fisica and Unita` di Ricerca INFM, UniVersita` della Calabria, 87036 ArcaVacata di Rende, Italy

C. La Rosa, D. Milardi, and D. Grasso Dipartimento di Scienze Chimiche, UniVersita` di Catania, Viale A. Doria 6, 95125 Catania, Italy ReceiVed: July 25, 1997; In Final Form: October 2, 1997

The thermal unfolding of azurin in D2O has been investigated by differential scanning calorimetry, optical density measurements, and electron paramagnetic resonance spectroscopy. The study has allowed us to relate the local conformational changes occurring around the active site of azurin with the unfolding of the whole protein as the temperature increases. DSC and OD experiments have shown that the thermal unfolding is, on the whole, irreversible and kinetically controlled. Moreover, by extrapolation of both the experimental heat capacity and the optical density data to infinite heating rate, we have separated the reversible step from the irreversible, kinetically controlled one and calculated the thermodynamic parameters of the time-independent part of the denaturation process. The whole of the results suggest that the unfolding of azurin in D2O follows the same pathway as observed in H2O (La Rosa et al. J. Phys. Chem. 1995, 99, 14864-14870),1 but it is shifted to a higher temperature. From the comparison of the thermodynamic unfolding parameters obtained in the two solvents, it results that D2O destabilizes the native state of the protein. According to an analysis of the thermodynamic behavior of model compounds in heavy water, this destabilizing effect can be mainly ascribed to the apolar groups of the protein. In addition, the region around the active site is enthalpically less influenced by changing the solvent with respect to the global protein. This behavior has been ascribed to the different solvent-azurin interactions in heavy and in light water. Finally, EPR results show that during the thermal unfolding, the active-site geometry changes from trigonal bipyramidal to square planar. Such a conformational change is not influenced by solvent isotopic effects.

Introduction Among the interactions stabilizing the native protein structure, protein-solvent interaction certainly plays a crucial role. It is known that hydration water is determinant in the activation of the protein functionality2,3 and that changes in the hydration solvent properties are critical in the process of both protein folding and stability. Information on the stability of a protein structure can be obtained by studying its denaturation, i.e., the transition from an ordered to a disordered state.4 This transition can be induced by changing different properties of the protein solution such as temperature, pH, pressure, and concentration of denaturants.5 The investigation of the effects induced by these external perturbations provides valuable information about the role of the solvent in maintaining the native structure. In a previous paper we have applied the generality of these concepts to the investigation of the thermal behavior of azurin in aqueous solution.1 For its peculiar spectroscopic features, this enzyme has been the focal point of intense structural researches over the past few years.6-11 However, so far, very little information is available on its thermal stability.12-14 Further interest in the study of the thermodynamics of this β-sheet protein comes out also from the fact that not many proteins of this class have been studied before.15 * To whom correspondence should be addressed. Fax number: ++39.984.493187. E-mail:[email protected].

The results of our studies in H2O1 have shown that azurin is a very stable protein, since temperatures higher than 70 °C are required for the irreversible denaturation. The irreversibility of the thermal transition of azurin makes both the classical thermodynamic analysis and the application of the statisticalmechanical deconvolution methods impossible. The model previously proposed describes the thermal denaturation path of azurin in aqueous solution in terms of two contributions: a reversible endothermic contribution, related to the disruption of the tertiary and secondary structure, followed by an irreversible exothermic one, which has been assigned to the aggregation of the unfolded polypeptidic chains.1,14 A mathematical extrapolation procedure of DSC data to infinite scanning rate was the key to the separation of the two effects, allowing us to calculate the thermodynamic parameters of the reversible process. The details about the procedure applied and the conditions for its applicability are given in ref 1. To obtain a better understanding of the thermodynamics of the interaction of water with azurin, solvent perturbation experiments are very helpful. The mildest perturbant of light water is its isotopic form D2O. In this paper we report on the thermal behavior of azurin in D2O, and the results are compared to the corresponding ones in H2O. The choice of this solvent arises also from the observation that although D2O and H2O can be viewed as forming essentially the same liquid, their effect on living systems is strikingly different. In fact, although all biological molecules

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1022 J. Phys. Chem. B, Vol. 102, No. 6, 1998 operate in aqueous solution, D2O is toxic, even lethal at high concentrations to higher animals and plants.16 Since there is a little reason to believe that large structural differences exist in a hydrogenated or deuterated protein,17 the understanding of the above effect, of course, must come from the understanding of the interaction of D2O with polypetides and proteins. Small differences between the two solvents do exist in the magnitude of some physical properties, such as freezing and boiling temperatures and dielectric constant.18 Moreover, various experimental and theoretical studies suggest that hydrophobic interactions are stronger in D2O than in H2O.19-21 This enhancement of hydrophobicity in D2O can provide an effective tool for the investigation of the role of the hydrophobic effects on the thermal behavior of a protein. On such a background, in this paper we use differential scanning calorimetry (DSC), optical density (OD) measurements, and electron paramagnetic resonance (EPR) spectroscopy to investigate the azurin/D2O system. DSC provides direct determination of the properties of the thermally induced structural transitions of azurin.22 Moreover, to relate the global conformational changes to those occurring within the copper environment, we also follow the optical density at λ ) 625 nm as a function of temperature under the same experimental conditions as the DSC ones. EPR is also used to characterize the copper active-site geometry in the native and final denaturated state. The results show that the isotopic effect is mainly evidenced by a lower stability of azurin at room temperature, while the unfolding pathway does not depend on the solvent. Experimental Section Azurin from Pseudomonas aeruginosa (MW of 14 000) was obtained from Sigma Chemical Co (St. Louis) and used without further purification. Potassium phosphate (analitical grade) was obtained from Fluka Chemie AG (Buchs, Switzerland). Heavy water (99.9%) was also from Sigma. DSC scans were carried out with a third-generation SETARAM (Lyon, France) microdifferential scanning calorimeter (microDSC III) with stainless steel 1-mL sample cells and the CS32 control unit interfaced to a personal computer. This instrument has a temperature resolution of 0.01 °C and a sensitivity of 90 µV/mW. The protein, at a concentration of 7 × 10-5 M, was dissolved in the appropriate 10 mM buffer. In particular, the solvents with different pD value (pD ) pHmeter + 0.4)23 have been obtained by using the following buffers: glycine/DCl (pD 2.5-3.0), sodium acetate/DCl (pD 3.9-4.5), and sodium phosphate/DCl (pD 6.8-7.4). The ionic strength was adjusted to 0.1 with sodium chloride. For each pD value, both the sample and the reference were scanned from 30 to 100 °C with a precision of (0.02 °C at scanning rates of 0.3, 0.5, 0.7, and 1.0 °C/min. Experiments with protein concentration ranging from 0.6 to 1.5 mg/mL have shown that DSC transitions do not depend on protein concentration (see the inset of Figure 1A). To obtain the excess heat capacity (Cp,exc) curves, bufferbuffer baselines were obtained at the same scanning rate and then subtracted from samples curves. All the Cp,exc curves were obtained using a fourth-order polynomial fit as the baseline as reported in Figure 1A. The average level of noise was about (0.4 µW, and the reproducibility at refilling was about 0.1 mJ K-1 mL-1. Calibration in energy was obtained by giving a definite power supply electrically generated by an EJ2 SETARAM joule calibrator within the sample cell. Optical density (OD) measurements were carried out with a JASCO 7850 spectrophotometer equipped with a Peltier-type

Guzzi et al.

Figure 1. (A) DSC thermogram of azurin in D2O after subtraction of buffer-buffer baseline. Protein concentration is 1.05 mg/mL, pD ) 7.03, ionic strength is 0.1 in NaCl, and scan rate is 0.5 °C/min. The baseline (dashed curve) was obtained as described in the text. The inset shows the Tmax dependence on the protein concentration. (B) Normalized OD625/temperature variation of 4.7 × 10-5 M azurin in D2O, pD ) 7.03, at a scan rate of 0.5 °C/min.

thermostated cell holder, model EHC-441, and a temperature programmer, model TPU-436 (precision (0.02 °C). Quartz cuvettes with a 1-cm optical path were used throughout. The experiments on azurin, 4.7 × 10-5 M, in deuterim oxide buffer solution, pD ) 7.03, were started 3 min after samples were positioned in the thermostated sample holder at an initial temperature of 50 °C. The temperature of the sample was measured directly by a YSI precision thermistor in the reference cuvette. The scanning rates were the same as used in the DSC measurements. Electron paramagnetic resonance (EPR) measurements on azurin, 1.7 × 10-4 M, in deuterim oxide buffer solution, pD ) 7.03, were carried out with a Bruker ER 200D-SRC X-band spectrometer equipped with an ESP 1600 data system. The EPR spectra were recorded at 77 K by plunging the sample solution in a finger Dewar containing liquid nitrogen. It is worthwhile to point out that the different experimental techniques used required a different protein concentration in order to obtain an acceptable signal-to-noise ratio. However, no concentration dependence of the experimental data has been observed. The reproducibility of all data presented in this work has been tested by repeating the measurements at least three times. Results and Discussion Differential Scanning Calorimetry. In Figure 1A the calorimetric profile of azurin in D2O, pD ) 7.03, in the temperature range 60-100 °C is shown. The DSC profile shows an intense heat absorption at Tmax ) 85.26 °C and an exothermic contribution at temperatures higher than 87 °C. The

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Figure 2. Effect of scanning rate on the excess-heat-capacity function of azurin in D2O: (a) 0.3, (b) 0.5, (c) 0.7, and (d) 1 °C/min. The protein concentration was 1.05 mg/mL in all experiments. pD ) 7.03.

TABLE 1: Effect of Scanning Rate on Temperatures Tt and Tmax of Azurin in Deuterium Oxide and in Aqueous Solution As Determined by OD and DSC, and Experimental ∆H Values V (°C/min)

Tt (°C)a

0.3 0.5 0.7 1

83.25 ( 0.08 83.85 ( 0.08 84.36 ( 0.08 85.65 ( 0.05

D2O 84.29 ( 0.03 85.26 ( 0.04 85.64 ( 0.06 86.29 ( 0.04

292.1 ( 19 356.3 ( 24 366.0 ( 26 456.2 ( 28

0.3 0.5 0.7 1

78.61 ( 0.05 c 80.03 ( 0.04 80.92 ( 0.06

H2Ob 81.02 ( 0.08 83.31 ( 0.07 84.44 ( 0.04 84.72 ( 0.05

276.7 ( 20 409.6 ( 22 478.6 ( 29 512.3 ( 21

Tmax (°C)a

∆H (kJ/mol)a

a Values expressed as mean ( standard deviation. b Data from La Rosa et al., 1995. c Not determined.

quite high Tmax value can be ascribed to a number of structural factors, including an intramolecular hydrogen-bond network, hydrophobic effects, and stabilization of the tertiary structure by the Cu2+ ion in the active site. The disulfide bridge is believed to play an important role too.24 No calorimetric reversibility was observed; i.e., a second DSC run of a previously scanned sample did not show any endothermic peak. This means that the thermal unfolding of azurin is, on the whole, irreversible so that it cannot be analyzed in light of classical thermodynamics. Since the thermal transition is irreversible, it is necessary to establish whether the reaction is under kinetic control and whether there is a change of molecularity during the heating of the solution.25,26 DSC scans at different scan rates allow us to determine whether the process is under kinetic control. On the other hand, the absence of an evident protein concentration effect on the shape and maximum (inset of Figure 1A) of the calorimetric curves excludes any change of molecularity during the unfolding. Figure 2 shows the Cp,exc profiles of azurin in D2O, pD ) 7.03, obtained at different scan rates (from 0.3 to 1 °C/min) in the 76-96 °C temperature range. It is noteworthy that Tmax increases by increasing the scan rate. From the Tmax values reported in Table 1 it is possible to calculate the apparent activation energy Eapp of the unfolding process of the whole protein molecule by using the following equation:26,27

( )

ln

V

Tmax2

)C-

Eapp RTmax

(1)

where Tmax is the temperature of the maximum heat absorption,

V (°C/min) is the scan rate, and C is a constant. From the slope of the linear plot of ln(V/Tmax2) vs 1/Tmax we have calculated the Eapp of the process, which is 625 ( 32 kJ/mol. Moreover, by looking at the exothermic peak localized at the end of the DSC transition (Figure 2), it can be noted that its amplitude diminishes when the scan rate increases. As was previously shown by some of us,28 such a peak decrease indicates that the exothermic contribution is time-dependent. It is, therefore, reasonable to consider the reversible step separable from the irreversible one by means of extrapolation of the calorimetric curves to infinite scanning rate.25,29,30 The procedure of extrapolation of the Cp,exc curve at infinite scanning rate has been discussed in detail elsewhere.1 The results obtained for the reversible extrapolated Cp,exc profile of azurin in D2O at pD ) 7.03 at infinite scanning rate are listed in Table 2. All calorimetric data collected suggest that the denaturation path of azurin in D2O is

NSUwF

(a)

where N is the native, U the unfolded, and F the final state. The chemical equilibrium between N and U has to be always established. This scheme has already been used to describe the thermal denaturation of azurin in H2O.1 To determine the thermodynamic and the kinetic parameters of the two steps of this process, the equations developed by Sanchez-Ruiz25 have been used. The approach of Sanchez-Ruiz, which originates from the classical Lumry-Eyring model,31 considers the heat exchange associated with the final step U w F to be negligible. However, some of us28 have improved the Sanchez-Ruiz model for the analysis of a process of type a when the enthalpy of the process U w F, ∆HF, is not negligible. Thus, the experimental curves, obtained at different scan rates (Figure 2), were simulated with the following equation:28

Cp,exc )

[

{

}

k ∆HU + + (K + 1) V RT2 K∆HU

2

] {

}

dT ∫TTKkK +1

1 1 kK exp ∆HF VK+1 V

0

(2)

where R is the gas constant, K is the thermodynamic equilibrium constant associated with the reversible step

{

K ) exp -

(

)}

∆HU 1 1 R T T1/2

(3)

and k is the kinetic constant of the irreversible step

{ RE(T1 - T1′)}

k ) exp -

(4)

∆HU is the thermodynamic enthalpy variation associated with the N S U process, E is the activation energy of the irreversible step, ∆HF is the enthalpy change associated with the irreversible process, and T1/2 and T′ are the temperatures at which the thermodynamic equilibrium constant K and the kinetic constant k approach unity, respectively. From the fit the unknown parameters, ∆HF, E, and T′ have been obtained. As an example, in Figure 3 the comparison between the experimental heat capacity curve of azurin in deuterium oxide buffer solution pD )7.03 recorded at 0.5 °C/min (solid line) and the corresponding simulation (dashed line) is shown. The good agreement between the experimental and the simulated profiles suggests that the model proposed provides a reliable

1024 J. Phys. Chem. B, Vol. 102, No. 6, 1998

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TABLE 2: Extrapolated DSC and OD Data at Infinite Scan Rate and Activation Energies for Azurin in D2O (pD ) 7.03) and H2O DSC ∆H∞ (kJ/mol)

Eapp (kJ/mol)

86.46 ( 0.07

593 ( 37

625 ( 32

86.30 ( 0.06 a

OD

T∞max (°C)

624 ( 73

D2O H2Oa

356 ( 23

T∞t (°C)

∆H∞app (kJ/mol)

Ea (kJ/mol)

86.15 ( 0.05

566 ( 25

507 ( 38

81.48 ( 0.08

506 ( 28

451 ( 32

Data from La Rosa et al., 1995.

Figure 3. Comparison of the experimental Cp,exc profile of azurin in D2O, pD ) 7.03, recorded at 0.5 °C/min (solid line) with the simulated profile (dashed line) using eq 2. The fitted parameters are reported in Table 3.

TABLE 3: Thermodynamic and Kinetic Parameters of the Thermal Denaturation of Azurin in D2O Obtained from the Fitting of the Experimental DSC Profiles at Different Scan Rates (See Text for Details)a V (°C/min)

∆HU (kJ/mol)

T1/2 (°C)

∆HF (kJ/mol)

E (kJ/mol)

T′ (°C)

mb

δc

0.3 0.5 0.7 1

590 586 601 594

86.3 86.5 86.4 86.5

-293 -238 -230 -131

591 483 364 424

88.9 88.5 89.2 86.9

4.1 3.8 3.7 2.8

19.7 17.7 21.5 14.9

a The minimum increments in the minimization procedure are 1 kJ/ mol for the enthalpies and 0.1 °C for the temperatures. The starting parameters are ∆HU ) 593 kJ/mol (range of (37 kJ/mol), T1/2 ) 86.4 °C (range of (0.2 °C). The kinetic parameters are free-floating. b m (kJ K-1 mol-1) is a measure of the accuracy of the fitting operation. It i i i is defined as m ) |∑i(Cp,theo - Cp,exc )/n|, where Cp,exc is the ith value i of the experimental Cp thermogram, Cp,theo is the corresponding calculated value, and n is the total number of points of the scan. c δ is i i the standard deviation of the Cp,theo - Cp,exc function calculated in the denaturation range.

description of the thermal denaturation of azurin. All the parameters used in the simulations are reported in Table 3. The different kinetic values found at different scan rates are ascribable to the fact that the final state, since it is obtained in an irreversible way, depends on the scan rate.1 Optical Density. The active site-bound copper is a suitable built-in probe useful for obtaining information on the copperenvironment changes during the thermal denaturation of azurin.14 In fact, since the copper ion is located in a hydrophobic region of the protein, any significant change in tertiary or secondary structure would be expected to alter the properties of the copper center and, in turn, the optical absorption, centered at λmax ) 625 nm, assigned to the charge-transfer (CT) band πS(Cys) f dx2-y2 (Cu2+).10 Figure 1B shows the normalized optical density variation at 625 nm in the temperature range 60-94 °C (OD625/T) of azurin

Figure 4. Normalized OD625/T profiles of azurin in D2O obtained at 0.3 (4), 0.5 (2), 0.7 (O), and 1 (b) °C/min.

in deuterium oxide buffer solution, pD)7.03, at a scan rate of 0.5 °C/min. The optical denaturation profile is typical of a single cooperative transition with a transition temperature Tt ) 83.85 °C defined as the midpoint of the OD transition. When the denaturation is complete (T > 87 °C), the protein solution appears bleached; the CT band is not recovered after cooling to room temperature. The loss of the blue color is ascribed to conformational changes of the azurin tertiary structure that permanently alter the relative position of the copper ion-ligand atoms. The disappearance of the CT band is not due to the oxidation of the Cys-112 at high temperature. In fact, the optical investigation of azurin under nitrogen atmosphere (not shown) gives the same results as in the presence of oxygen. To investigate the dependence of the OD625/T profile of azurin in D2O on the scan rate, measurements at 0.3, 0.5, 0.7, and 1.0 °C/min have been performed. The results are shown in Figure 4. As can be seen, the transition temperature Tt increases with the scan rate (Table 1). As previously shown, from the scanning-rate effect, it is possible to calculate the apparent activation energy Ea of this process using eq 1, where Eapp and Tmax are now substituted by Ea and Tt. From the slope of the linear plot ln(V/Tt2) vs 1/Tt an Ea value of 507 ( 38 kJ/mol is derived. The OD625/T profiles are, like the DSC ones, irreversible and time-dependent, so an experimental calculation of the thermodynamic parameters related to the active-site breakup with an increase in temperature is not allowed. However, by considering that the optical absorbance is reversible up to 78 °C, the existence of a reversible state can also be hypothesized for the thermal transition as revealed by optical spectroscopy. This assumption is supported by the observations that the overall three-dimensional features of azurin up to about 78 °C are indistinguishable from those of the native state, as FTIR spectroscopy13 and calorimetric data obtained up to 78 °C show.1,14 However, a local conformational transition that allows a variation of the Cu-S bond length, evidenced by the optical absorb-

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Figure 5. OD625/T profiles of azurin in (O) D2O and in (b) H2O obtained by extrapolation of the OD curves at infinite scan rate.

ance decreases, occurs. This conformational transition is reversible. The subsequent step (that occurs after 78 °C) corresponds to the breakup of the Cu-S bond. This last process is irreversible. These considerations allow us to approximate the optical transition as a sum of two effects, the first reversible and the second irreversible. In this respect, we have applied the extrapolation procedure at infinite scan rate to the experimental OD625/T curves obtained at the different scan rates. For each temperature T ) Ti, where 60 < Ti < 90 °C, the four OD values have been plotted as a function of 1/V. The experimental points have been fitted with a linear function, and the intercept with the y axis gives the OD value at infinite scan rate. This procedure has been applied to the OD625/T curves of azurin in D2O and H2O (last set of data from ref 1). The results are shown in Figure 5. From these extrapolated reversible OD curves the apparent enthalpy ∆H∞app has been calculated using the equation32

∆H∞app ) 4RT2∞ t

( ) ∂fU ∂T

Figure 6. Normalized OD625/time-decay curves of azurin in D2O at temperatures of (2) 81, (4) 80, (b) 79, and (O) 78 °C. The continuous lines represent the best fits of the experimental points according to eq 7.

(5)

Figure 7. EPR spectra at T ) 77 K of azurin in D2O in the (a) native and (b) final states.

where T∞t is the transition temperature of the extrapolated reversible curve and fU is

energy of azurin in light water (Ea) 441 ( 29 kJ/mol).14 This difference can be explained in terms of the relative strength of the hydrophobic interactions, which are stronger in D2O than in H2O.33 Electron Paramagnetic Resonance. The EPR spectrum of native azurin in D2O recorded at 77 K is shown in Figure 7a. The spectral features are typical of a type I copper ion with axial symmetry characterized by four hyperfine lines centered at g| and separated by A| and by a single, more intense resonance line centered at g⊥ at higher fields.6,34 The values of these magnetic parameters, which give information about the geometry and the atoms of the first coordination sphere of copper ion, are g| ) 2.273, g⊥ ) 2.051, and A| ) 55 G. Figure 7b shows the EPR spectrum of azurin in D2O at 77 K recorded after the protein solution has been heated at 90 °C for 10 min. After this time the solution appears bleached, indicating that the thermal denaturation was completed. The EPR parameters of this copper complex are g| ) 2.281, g⊥ ) 2.044, and A| ) 160 G. These values indicate that the copper geometry changes from tetrahedral to square-planar as the temperature increases beyond the transition temperature. In addition, the A| and g| values suggest that the two sulfur copper ligands are probably substituted from two oxygen atoms.35,36 This result is in agreement with that observed for the denaturation of azurin in H2O1 and suggests that the copper environment in the final state shows the same structural features in the two solvents.

fU )

T)T∞t

OD(T) - ODN(T) ODU(T) - ODN(T)

(6)

where OD(T) is the actual optical density and ODN and ODU are the OD values of the native and reversible states, respectively. The ∆H∞app values for azurin in D2O and in H2O and the corresponding T∞t values are reported in Table 2. The kinetics of the thermal breakup of the active site of azurin has also been studied by following the time-dependent OD625 variation at different temperatures T < Tt. First-order rate constants k have been calculated by fitting the experimental OD625/time profiles with the following equation:26

OD625(t) ) OD625(∞) + [OD625(0) - OD625(∞)] e-kt

(7)

where OD625(0) and OD625(∞) are the optical densities at t ) 0 and t ) ∞, respectively (Figure 6). The k values derived have been then used to calculate the apparent activation energy by using the Arrhenius equation ln k ) C - Ea/(RT). From the corresponding plot of ln k vs 1/T, an Ea value of 482 ( 33 kJ/ mol was calculated. As can be seen, there is a remarkable agreement between the values calculated using the two different methods, but both values are higher than the apparent activation

1026 J. Phys. Chem. B, Vol. 102, No. 6, 1998

Guzzi et al. the plot of ∆HU as a function of the transition temperature for several pD values is shown. The slope of the linear best fit of ∆HU vs T1/2, which represents the heat-capacity changes upon unfolding, gives 9.4 kJ K-1 mol-1 for azurin in D2O. This ∆Cp value, together with ∆HU and T1/2, allows us to calculate the thermodynamic functions of azurin in D2O using the following equations:

∆H(T) ) ∆HU - ∆Cp(T1/2 - T) ∆S(T) ) Figure 8. Temperature dependence of the extrapolated unfolding enthalpies ∆HU at different pD values. The solid line represents the linear fit of the data obtained as reported in the text. Its slope gives 9.4 kJ K-1 mol-1 for the heat-capacity change ∆Cp upon unfolding of azurin in D2O.

Solvent Isotope Effects on the Thermal Behavior of Azurin. The comparison of the thermal unfolding of azurin in D2O and H2O gives important information about the character of the protein-solvent interactions. From the extrapolated DSC data of azurin thermal unfolding in D2O and H2O (Table 2) it can be noted that the unfolding temperature T∞max obtained in D2O is slightly higher than that in H2O, in agreement with other studies carried out on different proteins.23,37,38 However, the extrapolated unfolding temperatures T∞t from OD data are noticeably different in the two solvents. The higher value found in D2O suggests that the copper environment gains stability in D2O compared with the environment in H2O. From the comparison of the DSC and OD extrapolated data, it can be noted that the protein behaves differently in the two solvents when observed in its overall properties or if a limited region of the protein (copper environment) is considered. In particular, in H2O the unfolding of the whole protein, observed by DSC, occurs after the “disruption” of the active site, whereas in D2O these two events are simultaneous (see Table 2). These results may be related to some specific interactions of D2O within the copper environment. A decrease in the unfolding enthalpy is also registered as an effect of thermal transition of azurin in D2O. This result is in agreement with the recent data reported by Makhatadze et al.38 on the isotopic effect on the thermal stability of RNase, lysozyme, and cytochrome c. To get more insight into the thermal unfolding of azurin in D2O and H2O, a comparison of the Gibbs free energy ∆G in the two solvents is necessary. ∆G depends on ∆ΗU, T1/2, and ∆Cp. Although the first two quantities have already been calculated, the irreversibility of the thermal denaturation of azurin prevents the experimental determination of ∆Cp ) CpUexc - CpNexc, (see ref 1 for details). A further complication comes from the fact that the method proposed by Murphy and Gill39 to calculate ∆Cp cannot be applied in the present case because the specific ∆Cp(-CONH-) and ∆Cp(-CH-) values related to the specific ∆Cp contribution of the peptide bonds and of the apolar hydrogen atoms are not available for D2O. ∆Cp was then calculated from the temperature dependence of the extrapolated ∆HU value. The ∆HU and the T1/2 relative to the thermally induced unfolding of azurin at several pD values have been calculated according to the extrapolation procedure at infinite scanning rate.1 In fact, even if the exothermic peak decreases at low pD, the irreversibility was still maintained. In Figure 8

∆G(T) ) ∆HU

T1/2 ∆HU - ∆Cp ln T1/2 T

(8) (9)

T1/2 - T T1/2 - ∆Cp(T1/2 - T) + T∆Cp ln T T (10)

which are plotted in Figure 9. In the same figure, the previously calculated thermodynamic functions of azurin in H2O1 are also shown. The behavior of the unfolding enthalpy and entropy changes of azurin in D2O are similar (see parts a and b of Figure). Both functions have lower values compared with the corresponding ones in H2O in the whole temperature range investigated. However, the difference between the unfolding parameters in the two solvents decreases with temperature and at T) 120 °C vanishes. This behavior is reasonable, since the chemicalphysical differences between H2O and D2O disappear at high temperatures.19 In principle, the difference in the enthalpy of unfolding of azurin in heavy and in light water can be expressed as the sum of three terms38-40 that correspond to (1) hydrogen exchange (replacement of exchangeable hydrogen atoms by deuterium atoms), (2) differences in the light and heavy water hydration properties of polar and apolar groups, and (3) other changes in the structures of the protein that occur as a consequence of effects 1 and 2. An estimate of the contribution of each term needs some consideration. The H/D exchange taking place during the protein unfolding occurs with different rates (it covers about 7 orders of magnitude).41,42 Although the higher exchange rate constants are attributed to exchangeable sites with a higher flexibility,41 the backbone protons have been found to be protected from exchange until the protein is perturbed by denaturation.43 This last contribution to the unfolding enthalpy has been shown to be negligible.44 On the other hand, comparative FT-IR investigations on azurin in H2O and D2O indicate that insignificant H f D exchange occurs at room temperature within the β strands.13 Finally, it has been also shown that, from a structural point of view, hydrogenated and deuterated proteins have very similar three-dimensional structures.17 The above considerations suggest that the observed difference ∆∆H ) ∆H(D2O) - ∆H(H2O) may be mainly assigned to the different hydration properties of polar and apolar groups of the protein in heavy and light water. In Figure 9c the ∆G functions for azurin in D2O and H2O are compared too. As can be noted, the ∆G function of azurin in D2O shows a lower value with respect to that of H2O, although this difference decreases with temperature and at about 80 °C is zero. In particular, the reduction of ∆G in D2O at the temperature of maximum stability is about 14 kJ/mol. This reduction observed at low temperature suggests that the isotopic substitution mainly destabilizes the native state of the protein.

Thermodynamics of Azurin

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Figure 10. Dependence of the enthalpy of transfer from H2O to D2O (∆Ht) measured at 25 °C for alcohols (9), amino acids (b), and ketones (2) on the number of apolar hydrogens (NCH) of these compounds. The best-fit slopes of the lines are -0.162 ( 0.015, -0.133 ( 0.029, and -0.180 ( 0.010 kJ mol-1 NCH-1, respectively. The average slope value is -0.158 kJ mol-1 NCH-1.

Figure 11. Dependence of the entropy of transfer from H2O to D2O (∆St) at 25 °C for some hydrocarbons on the number of apolar hydrogens (NCH) of these compounds. The slope of the line is -0.516 ( 0.120 J K-1 mol-1 NCH-1.

Figure 9. Thermodynamic denaturational functions ∆H(T) (a), ∆S(T) (b), and ∆G(T) (c) relative to the thermal denaturation of azurin in heavy and in light water.

More insight on the origin of the observed differences in the enthalpy and entropy of the unfolding of proteins in heavy and light water may be suggested from an analysis of the transfer of model compounds from H2O to D2O. Three different types of compounds (alcohols,33,45 amino acids,38 and ketones46) have been extensively studied, and their enthalpies of transfer (∆Ht) from H2O to D2O have been measured at 25 °C. The plot of ∆Ht as a function of the apolar hydrogen atoms NCH shown in Figure 10 reveals two significant features. The first is a linear dependence of ∆Ht on NCH, which means an additivity of the enthalpy of transfer. The second one is that the slopes of the best fits are not very different, with a deviation of only 20% from the average value of -0.158 kJ mol-1 NCH-1. This value represents the enthalpic contribution of one apolar hydrogen atom upon the transfer from H2O to D2O, regardless of the type of compound. Since in azurin NCH ) 757, the contribution to ∆∆H ascribable only to the difference in the hydrophobic effect

should be -119.61 kJ/mol. On the other hand, according to eq 8 the ∆∆H calculated for azurin at 25 °C is -89 kJ/mol. However, in general only 60-79% of the protein apolar surface is buried.47 By use of the Murphy and Gill39 model, it turns out that for a protein of 128 residues, like azurin, the fraction of buried apolar surface is 0.664. Taking into account this value, the hydrophobic contribution to ∆∆H (25 °C) is 0.664 × (-119.6) ) -79 kJ/mol. The difference between the total and the hydrophobic contributions to ∆∆H is ascribable to the hydration of the polar surface. In a similar way, the entropic contribution of the apolar hydrogens to the entropy of transfer ∆St from H2O to D2O can be estimated from model-compounds data. In Figure 11 is shown the linear plot of ∆St as a function of NCH of a series of hydrocarbons.20 The slope of this best fit, which is -0.516 J K-1 mol-1 NCH-1, represents the contribution of each apolar hydrogen atom to ∆St at 25 °C. If we consider the fraction of buried apolar hydrogen atoms, the apolar contribution to the entropy of transfer ∆∆S ) ∆S(D2O) - ∆S(H2O) of azurin at 25 °C is 757 × (-0.516) × 0.664 ) -0.259 kJ K-1 mol-1. If the same quantity is calculated by using eq 9, we found a value of -0.261 kJ K-1 mol-1, which is in excellent agreement with the calculated data. This means that the polar groups, from an entropic point of view, have no effect when deuterated water is used instead of light water. Finally, another difference between the thermal behavior of azurin in D2O and that in H2O can be observed from the general

1028 J. Phys. Chem. B, Vol. 102, No. 6, 1998 increases of the apparent activation energies in the presence of heavy water (Table 2). This result means that the irreversible step, which is ascribed to the polypeptide-chains interaction in the unfolded state, is slackened by D2O. The explanation for this effect is not trivial. In fact, its molecular origin can be ascribed to a sum of factors; the difference of the dielectric constants, and viscosity and hydrophobic interactions in the two solvents are likely to be responsible for this different kinetic behavior. Moreover, the more marked increase in Eapp with respect to Ea suggests that the phenomenon, observed by OD measurement and described by the Ea parameter, is less sensitive to the change of the solvent. This result is not surprising considering that OD data are related to the azurin copper site, which is located in a hydrophobic pocket where access to the solvent molecules is prevented. Concluding Remarks The combined use of DSC, OD, and EPR techniques allowed us to present a different thermal behavior of the global protein and of its active site. In particular, in H2O the copperenvironment changes occur before the unfolding of the overall protein. Such a difference vanishes in D2O. This result suggests that different hydrophobic interactions “modulate” the differences between the copper region and the overall protein. A comparison of the ∆G function in D2O and H2O has indicated that D2O destabilizes the native state of azurin at lower temperatures. This destabilizing effect, which has been mainly ascribed to hydrophobic interactions, has an enthalpic character. On the other hand, from an entropic point of view, the presence of D2O plays a stabilizing effect. This would suggest that, when exposed to the solvent, the hydrophobic groups may induce a higher degree of order in light water than in heavy water. This result is in agreement with previously obtained experimental data on model compounds.20 The results presented in this paper have some implications for the study of protein stability in general, and of azurin in particular. (i) Because of the headgroup effect, it is not always straightforward to extrapolate from the behavior observed with model compounds the one observed in proteins. Notwithstanding, model compounds are very helpful for elucidating, at least to a first approximation, the relative contributions of hydrophobic or hydrophilic components to the overall thermodynamic stability of the protein. (ii) Hydration effects are, concerning their hydrophobic part, additive; that is, they can be scaled with the number of apolar hydrogens of the protein. (iii) The study of protein stability requires an analysis of its thermodynamics not only in terms of the Gibbs energy but also in terms of enthalpy and entropy, since the enthalpy-entropy compensation taking place in aqueous solution may obscure important details of the overall energetics of the protein. Acknowledgment. One of us (R.G.) thanks the University of Calabria for a postdoctoral fellowship. This work has been supported by MURST (Ministero dell’ Universita` e della Ricerca Scientifica e Tecnologica), CNR (Consiglio Nazionale delle Ricerche), INFM (Istituto Nazionale di Fisica della Materia), and CIB (Consorzio Interuniversitario Biotecnologie). References and Notes (1) La Rosa, C.; Milardi, D.; Grasso, D.; Guzzi, R.; Sportelli, L. J. Phys. Chem. 1995, 99, 14864-14870. (2) Rupley, J. A.; Careri, G. AdV. Protein Chem. 1991, 4, 37-172. (3) Steinbach, P. J.; Loncharich, R. J.; Brooks, B. R. Chem. Phys. 1991, 158, 383-394. (4) Privalov, P. L. Annu. ReV. Biophys. Chem. 1989, 18, 47-69.

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