Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron protein: preparation and study of Fe(II)-M121E azurin.
J Am Chem Soc
; 136(35): 12337-44, 2014 Sep 03.
Article
em En
| MEDLINE
| ID: mdl-25082811
ABSTRACT
Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild-type azurin (wtAz) with Glu and Fe(II), respectively. In contrast to the redox inactive Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pseudomonas aeruginosa
/
Azurina
/
Proteínas de Bactérias
/
Ferroproteínas não Heme
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
J Am Chem Soc
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Estados Unidos