In mammalian peroxidases the proximal histidine is in close interaction with a fully conserved asparagine which in turn is hydrogen bonded with an arginine that stabilizes the propionatesubstituent of pyrrol ring D in bent conformation. In order to probe the role of this rigid proximal architecture for structural integrity and catalysis of human myeloperoxidase (MPO), the variants Asn421Asp, Arg333Ala and Arg333Lys have been recombinantly expressed in HEK cell lines. The standard reduction potential of the Fe(III)/Fe(II) couple of Asn421Asp was still wild-type-like (-50 mV at pH 7.0) but the spectral properties of the ferric and ferrous forms as well as of higher oxidationstates showed significant differences. Additionally, rates of ligand binding and oxidation of both one and two-electron donors were diminished. The effect of exchange of Arg333 was even more dramatic. We did not succeed in production of mutant proteins that could bind heme at the active site. The importance of this His-Asn-Arg triad in linking the heme iron with the propionate at pyrrol ring D for heme insertion and binding as well as in maintenance of the architecture of the substrate bindingsite(s) at the entrance to the heme cavity is discussed.

Manipulating the proximal triad His-Asn-Arg in human myeloperoxidase / J., Stampler; Bellei, Marzia; M., Soudi; C., Gruber; Battistuzzi, Gianantonio; P. G., Furtmüller; C., Obinger. - In: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. - ISSN 0003-9861. - STAMPA. - 516:(2011), pp. 21-28. [10.1016/j.abb.2011.09.007]

Manipulating the proximal triad His-Asn-Arg in human myeloperoxidase

BELLEI, Marzia;BATTISTUZZI, Gianantonio;
2011-01-01

Abstract

In mammalian peroxidases the proximal histidine is in close interaction with a fully conserved asparagine which in turn is hydrogen bonded with an arginine that stabilizes the propionatesubstituent of pyrrol ring D in bent conformation. In order to probe the role of this rigid proximal architecture for structural integrity and catalysis of human myeloperoxidase (MPO), the variants Asn421Asp, Arg333Ala and Arg333Lys have been recombinantly expressed in HEK cell lines. The standard reduction potential of the Fe(III)/Fe(II) couple of Asn421Asp was still wild-type-like (-50 mV at pH 7.0) but the spectral properties of the ferric and ferrous forms as well as of higher oxidationstates showed significant differences. Additionally, rates of ligand binding and oxidation of both one and two-electron donors were diminished. The effect of exchange of Arg333 was even more dramatic. We did not succeed in production of mutant proteins that could bind heme at the active site. The importance of this His-Asn-Arg triad in linking the heme iron with the propionate at pyrrol ring D for heme insertion and binding as well as in maintenance of the architecture of the substrate bindingsite(s) at the entrance to the heme cavity is discussed.
516
21
28
Manipulating the proximal triad His-Asn-Arg in human myeloperoxidase / J., Stampler; Bellei, Marzia; M., Soudi; C., Gruber; Battistuzzi, Gianantonio; P. G., Furtmüller; C., Obinger. - In: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. - ISSN 0003-9861. - STAMPA. - 516:(2011), pp. 21-28. [10.1016/j.abb.2011.09.007]
J., Stampler; Bellei, Marzia; M., Soudi; C., Gruber; Battistuzzi, Gianantonio; P. G., Furtmüller; C., Obinger
File in questo prodotto:
File Dimensione Formato  
MPO_Prox_His_mut2011.pdf

non disponibili

Tipologia: Versione pubblicata dall'editore
Dimensione 1.44 MB
Formato Adobe PDF
1.44 MB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

Licenza Creative Commons
I metadati presenti in IRIS UNIMORE sono rilasciati con licenza Creative Commons CC0 1.0 Universal, mentre i file delle pubblicazioni sono rilasciati con licenza Attribuzione 4.0 Internazionale (CC BY 4.0), salvo diversa indicazione.
In caso di violazione di copyright, contattare Supporto Iris

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/667849
Citazioni
  • ???jsp.display-item.citation.pmc??? 2
  • Scopus 7
  • ???jsp.display-item.citation.isi??? 6
social impact