Oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering.
Iso-1-cytochrome c, as any other hemeprotein, is able to react with hydrogen peroxide and to engage in the peroxidase cycle. However, peroxidases are irreversibly inactivated by their substrate, hydrogen peroxide. The oxidative inactivation of hemeproteins is mechanism based and arises as the consequence of unproductive electron abstraction reactions. Protein elements, such as the porphyrin ring or the protein backbone, act as simultaneous and competing electron sources even in the presence of exogenous reducing substrates, leading to a decline in activity. It is hypothetically possible to alter the intramolecular electron transfer pathways by direct replacement of low redox potential residues around the active site; as a consequence, the inactivation process would be delayed or even suppressed. To demonstrate this hypothesis, a redox-inspired strategy was implemented until an iso-1-cytochrome c variant fully stable at catalytic concentrations of hydrogen peroxide was obtained. This variant, harboring the N52I,W59F,Y67F,K79A,F82G substitutions, preserved the catalytic performance of the parental protein but achieved a 15-fold higher total-turnover number. The phenotype of this variant was reflected in the stability of its electronic components, allowing identification of a protein-based radical intermediate mechanistically similar to Compound I of classical peroxidases. The results presented here clearly demonstrate that redox-inspired protein engineering is a useful tool for the rational modulation of intramolecular electron transfer networks.
Valderrama
Brenda
B
Department of Cellular Engineering and Biocatalysis, Biotechnology Institute, National University of Mexico, AP 510-3, Cuernavaca, México. brenda@ibt.unam.mx
García-Arellano
Humberto
H
Giansanti
Stefania
S
Baratto
M Camilla
MC
Pogni
Rebecca
R
Vazquez-Duhalt
Rafael
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
C014059:CYC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D006861:Hydrogen Peroxide