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Titolo:
Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies
Autore:
Perry, A; Lian, LY; Scrutton, NS;
Indirizzi:
Univ Leicester, Dept Biochem, Leicester LE1 7RH, Leics, England Univ Leicester Leicester Leics England LE1 7RH er LE1 7RH, Leics, England
Titolo Testata:
BIOCHEMICAL JOURNAL
, volume: 354, anno: 2001,
parte:, 1
pagine: 89 - 98
SICI:
0264-6021(20010215)354:<89:TROPOP>2.0.ZU;2-K
Fonte:
ISI
Lingua:
ENG
Soggetto:
AMINO-ACID-SEQUENCE; ARCHAEBACTERIUM PYROCOCCUS-FURIOSUS; ENZYMATIC OMEGA-OXIDATION; DESULFOVIBRIO-VULGARIS; CLOSTRIDIUM-PASTEURIANUM; SUBSTITUTED RUBREDOXIN; METAL SUBSTITUTION; 1.5-A RESOLUTION; ZINC-RUBREDOXINS; ALKBAC OPERON;
Keywords:
alkaline hydroxylation; electron transfer; metalloprotein;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
50
Recensione:
Indirizzi per estratti:
Indirizzo: Scrutton, NS Univ Leicester, Dept Biochem, Adrian Bldg,Univ Rd, Leicester LE1 7RH, Leics, England Univ Leicester Adrian Bldg,Univ Rd Leicester Leics England LE1 7RH
Citazione:
A. Perry et al., "Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies", BIOCHEM J, 354, 2001, pp. 89-98

Abstract

A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin: a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms afterprecipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively, In steady-state assays, therecombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c, The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci, 2, 1756-1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presenceof four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jorgensen's electronegativity theory, the 240 nm band is attributable to a CysS-Cd(II) charge transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However: the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into theputative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a findingconsistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 25/11/20 alle ore 07:16:33