title: Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase.
abstract: Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 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) approximately 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<-->U-->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.
authors.affiliation: Dipartimento di Fisica e Unità INFM, Laboratorio di Biofisica Molecolare, Università della Calabria, Ponte P. Bucci-Cubo 31C, I-87036, Arcavacata di Rende (CS), Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Stirpe
authors.forename: Rita
authors.initials: R
authors.lastname: Guzzi
authors.forename: Hein
authors.initials: H
authors.lastname: Wijma
authors.forename: Martin Ph
authors.initials: MP
authors.lastname: Verbeet
authors.forename: Gerard W
authors.initials: GW
authors.lastname: Canters
authors.forename: Luigi
authors.initials: L
authors.lastname: Sportelli
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D003300:Copper / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D006358:Hot Temperature; D009572:Nitrite Reductases / Q000737:chemistry*; D011489:Protein Denaturation; D013816:Thermodynamics
chemical_list: D003300:Copper; D009572:Nitrite Reductases