Cytochrome c Mutants for Superoxide Biosensors

Abstract

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.

The Anti-Apoptotic Bcl-xL Protein, a New Piece in the Puzzle of Cytochrome C Interactome

“…Full length human cytochrome c was expressed and purified as reported in the literature [36] in the unlabeled and 15 N-labeled form.…”

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

“…1A), Cc acts as a redox regulatory protein within the mitochondrial intermembrane space (10). In addition, Cc aids in controlling ROS levels by oxidizing superoxide anions (11) and exhibiting peroxidase activity (12); the latter, however, also yields lipid peroxidation (13,14). Furthermore, Cc plays a crucial role in programmed cell death, a process that is only partially understood (15)(16)(17)(18)(19)(20)(21).…”

Detection of Biomarkers for Different Diseases on Biosensor Surfaces Part II

“…In this chapter we analyze the binding and dissociation kinetics (if applicable) of (1) IFN-gamma as a function of aptamer variants and inclusion of spacer in addition to spacer (Tuleuova et al, 2010), (2) GST-N protein in PBS and GST-N protein in 10-fold diluted serum to an LPSCF fiber-optic biosensor (Huang et al, 2009), (3) cytochrome c mutant to a superoxide biosensor (Wegerich et al, 2009), (4) CA-II to an ABS ligand on an SPR biosensor surface (Williams et al, 2009), (5) glycerol secretion from differentiated (murine 3T3-L1) adipocytes to a microfluidic platform for fluorescence-based assay (Clark et al, 2010), and (6) different concentrations of CRP in solution to a sandwich-type assay using a label-free detection method, reflectometric interference spectroscopy (Albrecht et al, 2010).…”