Cytochrome C mutants for superoxide biosensors.
The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV-vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.
Wegerich
Franziska
F
Interfaces, Max Planck Institute of Colloids and Interfaces, 14476 Potsdam-Golm, Germany.
Turano
Paola
P
Allegrozzi
Marco
M
Möhwald
Helmuth
H
Lisdat
Fred
F
D015374:Biosensing Techniques / Q000379:methods*; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004563:Electrochemistry; D004566:Electrodes; D006418:Heme / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013481:Superoxides / Q000032:analysis* / Q000378:metabolism
D050505:Mutant Proteins; D013481:Superoxides; D006418:Heme; D045304:Cytochromes c; D007501:Iron