Many current anti-
cancer therapies rely on increasing the intracellular reactive
oxygen and
nitrogen species (RONS) contents with the aim to induce irreparable damage, which subsequently results in
tumor cell death. A novel tool in
cancer therapy is the use of cold atmospheric plasma (CAP), which has been found to be very effective in the treatment of many different
cancer cell types in vitro as well as in vivo, mainly through the vast generation of RONS. One of the key determinants of the cell's fate will be the interaction of RONS, generated by CAP, with important
proteins, i.e. redox-regulatory
proteins. One such
protein is
cytoglobin (CYGB), a recently discovered
globin proposed to be involved in the protection of the cell against oxidative stress. In this study, the effect of plasma-produced RONS on CYGB was investigated through the treatment of CYGB with CAP for different treatment times. Spectroscopic analysis of CYGB showed that although chemical modifications occur, its secondary structure remains intact. Mass spectrometry experiments identified these modifications as oxidations of mainly
sulfur-containing and
aromatic amino acids. With longer treatment time, the treatment was also found to induce nitration of the
heme. Furthermore, the two surface-exposed
cysteine residues of CYGB were oxidized upon treatment, leading to the formation of intermolecular
disulfide bridges, and potentially also intramolecular
disulfide bridges. In addition, molecular dynamics and docking simulations confirmed, and further show, that the formation of an intramolecular
disulfide bond, due to oxidative conditions, affects the CYGB 3D structure, thereby opening the access to the
heme group, through gate functioning of His117. Altogether, the results obtained in this study (1) show that plasma-produced RONS can extensively oxidize
proteins and (2) that the oxidation status of two redox-active cysteines lead to different conformations of CYGB.