14864 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA J. Phys. Chem. zyxwvut 1995, 99, zyxwvut 14864-14870 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM Thermodynamics of the Thermal Unfolding of Azurin Carmelo La Rosa,* Danilo Milardi, and Domenico Grasso Dipartimento di Scienze Chimiche, Universitb di Catania, V.le A. Doria, 6-95125 Catania, Italy Rita Guzzi and Luigi Sportelli Dipartimento di Fisica, Universitb della Calabria, 87036 Arcavacata di Rende, Cosenza, Italy Received: January 4, 1995; In Final Form: July 19, 1995@ The thermal denaturation of azurin from Pseudomonas aeruginosa was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR), and optical density (OD) experiments, with the aim of determining its thermodynamic stability and the thermally induced conformational changes of its active site. DSC experiments have shown an irreversible and complex unfolding path. In order to characterize the kinetically controlled step, DSC measurements were carried out at different scan rates. An extrapolation of the experimental heat capacity data to infinite scan rate allowed all the kinetic and thermodynamic parameters related to the process to be obtained. All these parameters extracted from the calorimetric data were verified by means of a curve-fitting program using an equation containing all information necessary to fully describe the unfolding process in details. Thermal denaturation, followed up to 82 "C by ESR and OD measurements, allowed us to study the structural variations of the copper environment at different temperatures. The AHLI thermodynamic, together with the value of AC, calculated according to an approach taking into account the common features of protein unfolding and dissolution of hydrophobic compounds, was used to evaluate the thermodynamic stability (AG) for the reversible process over the entire temperature range of denaturation. The high value of the maximum stability thus calculated was explained by the stabilizing effect of copper. Introduction Azurin is a small blue copper protein' that acts as an electron transfer agent in the redox systems of certain bacteria. Together with plastocyanin, azurin is the best characterized protein of this class. Recently, studies have been concentrated on the distinct spectroscopic properties of azurins and plastocyanins.2 These proteins exhibit a very intense absorption band in the visible region of the electromagnetic spectrum with 595 zyxwvuts < zyxwvu A,,,= < 630 nm and E FZ 500 M-' cm-I, unusually high redox potentials (240-400 mV), and a characteristic narrow hyperfine splitting in the ESR ~ p e c t r a . ~ . ~ X-ray diffraction studies on crystals of small blue copper proteins have led to the determi- nation of the high-resolution three-dimensional structures of various azurin~.~.~ The structure of azurin from Pseudomonas aeruginosa (P. aeruginosa) has also been p~blished.~.~ The thermodynamic stability of the tertiary structure of this enzyme has been the focal point of intense research over the past few years. In particular, previous works7 showed a structure which is highly resistant to thermal unfolding: tem- peratures exceeding 70 "C are necessary for irreversible unfolding. This unusual thermal resistance has been generally ascribed to a number of factors, including disulfide bridges, intramolecular hydrogen bonds, hydrophobic effects, and sta- bilization by Cu2+ bindi~~g.~-'O The whole of these effects can be detected by micro- differential scanning calorimetry (micro-DSC) measurements, when samples of enzyme in a suitable environment are temperature scanned.' The thermodynamic analysis of the calorimetric profiles is not directly possible because the thermal denaturation of azurin is irreversible. This irreversibility makes both the application of statistical-mechanical deconvolution methods' and classical thermodynamic analysis impossible. * To whom correspondence should be addressed. @Abstractpublished in zyxwvutsrqp Advance ACS Abstracts, September 15, 1995. 0022-365419512099- 14864$09.0010 The remarkable asymmetry of the DSC curves at the end of the transition is a further complication in the analysis of the unfolding process. It is ascribable both to exothermic phenom- ena and to the occurrence of kinetic factors.I3 So far, the thermodynamic analysis of the DSC curves of proteins showing calorimetric irreversibility has in general been considered impossible. However, in some cases, under specific conditions, even in the presence of calorimetric irreversibility, a thermodynamic analysis has been carried outsi4 In this paper we separate the effects associated with the reversible step of the denaturation process from those associated to the irreversible step, calculating the characteristic parameters in each case. These parameters were subsequently used to simulate the complete C,,,, profiles by means of a best fit program using the SIMPLEX minimization algorithm based on an equation previously developed on the basis of the hypoth- esized pathway of unfolding. The model used describes the denaturation path as the sum of two effects: an endothermic effect comprising the energy involved in the destruction of the protein's three-dimensional structure and an exothermic effect ascribable to the aggregation of the polypeptidic chain network. The first of these effects is, as will be explained, reversible, and in this case a thermodynamic analysis is possible. In contrast the second effect, which is assumed to be slow with respect to the reversible unfolding equilibrium, is irreversible and kinetically controlled. Optical density (OD) measurements, carried out in the same experimental conditions as the calorimetric ones, allowed us to calculate all kinetic variables associated with the transition involving the copper environment. The geometry of copper environment in the different states was investigated by means of electron spin resonance (ESR) experiments that made the correct interpretation of all the conformational changes reliable. zyxwvutsrqp 0 1995 American Chemical Society