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Thermal stability effects of removing the type-2 copper ligand His306 at the interface of nitrite reductase subunits

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Abstract

Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate, \({\text{N}}_{3} \overset k \longleftrightarrow {\text{U}}_{3} \xrightarrow{k}{\text{F}} \) (N—native, U—unfolded and F—final). To understand the role of interfacial residues on protein stability, a type-2 copper site ligand, His306, has been mutated to an alanine. The characterization of the native state of the mutated protein highlights that this mutation prevents copper ions from binding to the type-2 site and eliminates catalytic activity. No significant alteration of the geometry of the type-1 site is observed. Study of the thermal denaturation of this His306Ala NiR variant by differential scanning calorimetry shows an endothermic irreversible profile, with maximum heat absorption at T max ≈ 85°C, i.e., 15°C lower than the corresponding value found for wild-type protein. The reduction of the protein thermal stability induced by the His306Ala replacement was also shown by optical spectroscopy. The denaturation pathway of the variant is compatible with the kinetic model \({\text{N}}_{3} \xrightarrow{k}{\text{F}}_{3} , \) where the protein irreversibly passes from the native to the final state. No evidence of subunits’ dissociation has been found within the unfolding process. The results show that the type-2 copper sites, situated at the interface of two monomers, significantly contribute to both the stability and the denaturation mechanism of NiR.

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Authors and Affiliations

  1. Dipartimento di Fisica e Unità CNISM, Università della Calabria, Ponte P. Bucci, Cubo 31C, 87036, Arcavacata di Rende, Cosenza, Italy

    Andrea Stirpe, Luigi Sportelli & Rita Guzzi

  2. Gorleaus Laboratories, Metallo Protein Group, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands

    Hein Wijma & Martin Ph. Verbeet

Authors
  1. Andrea Stirpe
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  2. Luigi Sportelli
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  3. Hein Wijma
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  4. Martin Ph. Verbeet
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  5. Rita Guzzi
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Correspondence to Rita Guzzi.

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Stirpe, A., Sportelli, L., Wijma, H. et al. Thermal stability effects of removing the type-2 copper ligand His306 at the interface of nitrite reductase subunits. Eur Biophys J 36, 805–813 (2007). https://doi.org/10.1007/s00249-007-0151-5

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  • DOI: https://doi.org/10.1007/s00249-007-0151-5

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