Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type I is present in each subunit whereas type 2 is localized at the subunits... Show moreNitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type I is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T-m approximate to 100 degrees C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry-Eyring model, N double left right arrow U double right arrow F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed. (c) 2005 Elsevier B.V All rights reserved. Show less
Nitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover... Show moreNitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover must be quantitatively assessed. The enzyme nitrite reductase from Alcaligenes faecalis contains a redox-active blue copper centre and a nonblue enzyme-active copper centre. It can be covalently tethered to modified gold-electrode surfaces in configurations in which direct electron transfer is possible. A surface cysteine mutant of the enzyme can be similarly immobilised on bare electroactive gold substrates. Under such circumstances, however, electron transfer cannot be effectively coupled with substrate catalytic turnover. In using either the natural redox partner, pseudoazurin, or ruthenium hexammine as an "electron-shuttle" or "conduit" between enzyme and a peptide-modified electrode surface, the coupling of electron transfer to catalysis can be utilised in the development of an amperometric nitrite sensor. Show less
Maruccio, G.; Biasco, A.; Visconti, P.; Bramanti, A.; Pompa, P. P.; Calabi, F.; ... ; Canters, G. W. 2005
A protein field-effect transistor (Pro-FET) based on the blue-copper protein azurins (see Figure) and operating at room temperature and ambient pressure is demonstrated. The transfer... Show moreA protein field-effect transistor (Pro-FET) based on the blue-copper protein azurins (see Figure) and operating at room temperature and ambient pressure is demonstrated. The transfer characteristics of the Pro-FET exhibit a pronounced resonance due to the switch from behaving as a n-metal oxide semiconductor FET (n-MOSFET) to a p-MOSFET. Carrier transport through the device is explained in terms of an equilibrium between the two possible oxidation states of the redox site (Cu1+ and Cu2+). Show less
A comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning... Show moreA comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning calorimetry and optical spectroscopic techniques.For wild type plastocyanin the transition temperature, determined from the calorimetric profiles, is 62.7 degreesC at the scan rate of 60 degreesC/h, whereas for the mutant it is reduced to 58.0 degreesC. In both cases, the endothermic peak is followed by an exothermic one at higher temperatures.The unfolding process monitored by optical absorption at 596 nm also reveals a reduced thermal stability of the mutated plastocyanin compared to the wild type protein, with transition temperatures of 54.8 and 58.0 degreesC, respectively. For both proteins, the denaturation process was found to be irreversible and dependent on the scan rate preventing the thermodynamic analysis of the unfolding process.In parallel, small conformational changes between wild type and mutant plastocyanin emerge from fluorescence spectroscopy measurements. Here, a difference in the interaction of the two proteins between the microenvironment surrounding the fluorophores and the solvent was proposed.The destabilization observed in the disulfide containing mutant of plastocyanin suggests that the double mutation, Ile2lCys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink. (C) 2004 Elsevier B.V. All rights reserved. Show less
We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely... Show moreWe have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a “solid state protein film” maintains its nativelike conformation and ET function, even after removal of the aqueous solvent. Show less
Wijma, H.J.; Canters, G.W.; Vries, S. de; Verbeet, M.P. 2004
The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and... Show moreThe copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions of the catalyzed reaction in the pH range of 6-8. For this, nitric oxide was monitored by a Clark-type electrode, and the redox state of pseudoazurin was measured by optical spectroscopy. The equilibrium constant (K-eq) depends on the reduction potentials of pseudoazurin and nitrite/nitric oxide, both of which vary with pH. Above pH 6.2 the formation of NiR substrates (nitrite and reduced pseudoazurin) is favored over the products (NO and oxidized pseudoazurin). At pH 8 the K-eq amounts to 103. The results show that dissimilatory nitrite reductases catalyze an unfavorable reaction at physiological pH (pH = 7-8). Consequently, nitrous oxide production by copper-containing nitrite reductases is unlikely to occur in vivo with a native electron donor. With increasing pH, the rate and specificity constant of the forward reaction decrease and become lower than the rate of the reverse reaction. The opposite occurs for the rate of the reverse reaction; thus the catalytic bias for nitrite reduction decreases. At pH 6.0 the kat for nitrite reduction was determined to be 1.5 x 10(3) s(-1), and at pH 8 the rate of the reverse reaction is 125 s(-1). Show less
Andolfi, L.; Bruce, D.; Cannistraro, S.; Canters, G.W.; Davis, J.J.; Hill, H.A.O.; ... ; Astier, Y. 2004
Site-specifically engineered disulphide or surface cysteine residues have been introduced into two blue copper proteins, Pseudomonas aeruginosa azurin and Populus nigra plastocyanin, in order to... Show moreSite-specifically engineered disulphide or surface cysteine residues have been introduced into two blue copper proteins, Pseudomonas aeruginosa azurin and Populus nigra plastocyanin, in order to facilitate protein chemisorption on gold electrodes. The subsequently formed well-defined protein monolayers gave rise to robust electrochemical responses and electron transfer rates comparable to those observed at modified electrode surfaces. Proximal probe characterisation confirms the presence, at high coverage, of well-ordered protein adlayers. Additionally, gold-metalloprotein affinity is such that molecular-level tunnelling and topographic analyses can be carried out under aqueous solution. The approaches outlined in this work can, in principal, be extended to the generation of arrays of any redox-active biomolecule. (C) 2003 Elsevier B.V. All rights reserved. Show less
The assessment of the folding and of the structural stability of a protein in air, upon immobilization in the solid state, represents a critical point from both a fundamental point of view and for... Show moreThe assessment of the folding and of the structural stability of a protein in air, upon immobilization in the solid state, represents a critical point from both a fundamental point of view and for the development of solid state nanobioelectronics. The recent demonstrations by Rinaldi [R. Rinaldi , Adv. Mater. 14, 1453 (2002); Appl. Phys. Lett. 82, 472 (2003); Ann. (N.Y.) Acad. Sci. 1006, 187 (2003)] of protein-based solid state devices and transistors working in air have raised an intriguing question about the behavior of a biomolecule under nonphysiological conditions. The operation principle of the realized devices is based on the physiological electron transfer function of the metalloprotein azurin. This means that azurin should retain its shape and functionality also in the solid state when utilized in air and at room temperature. In this Brief Report, we prove this claim by analyzing the conformational state of the azurin monolayers developed for such devices by means of intrinsic fluorescence spectroscopy. We show that the immobilization of azurins in the solid state under nonliquid conditions, by means of a specific chemisorption process, does not necessarily lead to protein denaturation. This result is of great importance because it opens up interesting perspectives for the development of solid state hybrid nanodevices for electronic applications requiring nonliquid environments. Show less
Andolfi, L.; Canters, G.W.; Verbeet, M.P.; Cannistraro, S. 2004
The study of the electronic conduction through plastocyanin (PC) mutants assembled on a gold surface has been addressed by scanning tunneling spectroscopy. The two mutants exploit a single thiol... Show moreThe study of the electronic conduction through plastocyanin (PC) mutants assembled on a gold surface has been addressed by scanning tunneling spectroscopy. The two mutants exploit a single thiol group (PCSH) or a disulfide bridge (PCSS) to covalently bind at gold surface. The I-V measurements were performed by positioning the STM tip on top of a single molecule and sweeping the bias potential between +/- 1 V, under both ambient and controlled atmosphere. For PCSS, under ambient conditions, asymmetric I-V characteristics were obtained, which disappear under nitrogen atmosphere. PCSH, instead shows a symmetric I-V relation in air and under nitrogen environment. Here, as factors underlying this distinct electron conductive behaviour, a potential role for hydration water molecules and for copper redox levels are discussed. (C) 2003 Elsevier B.V. All rights reserved. Show less
We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely... Show moreWe have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a “solid state protein film” maintains its nativelike conformation and ET function, even after removal of the aqueous solvent. Show less
Alessandrini, A.; Gerunda, M.; Canters, G.W.; Verbeet, M.P.; Facci, P. 2003
Cu- and Zn-azurin chemisorbed on Au(111) have been comparatively investigated by electrochemical scanning tunnelling microscopy in buffer solution. Cu-azurin shows a marked tunnelling current... Show moreCu- and Zn-azurin chemisorbed on Au(111) have been comparatively investigated by electrochemical scanning tunnelling microscopy in buffer solution. Cu-azurin shows a marked tunnelling current resonance upon substrate potential at -0.21 V (vs SCE), whereas Zn counterparts do not. These data, discussed in terms of current theories on electron tunnelling through redox adsorbates, demonstrate the role of the electroactive metal ion present in the active site in assisting electron transfer via this metalloprotein, (C) 2003 Elsevier B.V. All rights reserved. Show less
We show that the electron-transfer protein azurin can be used to fabricate biomolecular rectifiers exploiting its native redox properties, chemisorption capability and electrostatic features. The... Show moreWe show that the electron-transfer protein azurin can be used to fabricate biomolecular rectifiers exploiting its native redox properties, chemisorption capability and electrostatic features. The devices consist of a protein layer interconnecting nanoscale electrodes fabricated by electron beam lithography. They exhibit a rectification ratio as large as 500 at 10 V, and operate at room temperature and in air. (C) 2003 American Institute of Physics. Show less
The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein ... Show moreThe disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (La Rosa et al. 1995. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the protein's overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin. Show less
