cofactor binding protects flavodoxin against oxidative stress代数余子式绑定保护flavodoxin对抗氧化应激.pdfVIP

Cofactor Binding Protects Flavodoxin against Oxidative Stress 1. 1. 2¤ 2 Simon Lindhoud , Willy A. M. van den Berg , Robert H. H. van den Heuvel , Albert J. R. Heck , 1 1 Carlo P. M. van Mierlo , Willem J. H. van Berkel * 1 Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands, 2 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Abstract In organisms, various protective mechanisms against oxidative damaging of proteins exist. Here, we show that cofactor binding is among these mechanisms, because flavin mononucleotide (FMN) protects Azotobacter vinelandii flavodoxin against hydrogen peroxide-induced oxidation. We identify an oxidation sensitive cysteine residue in a functionally important loop close to the cofactor, i.e., Cys69. Oxidative stress causes dimerization of apoflavodoxin (i.e., flavodoxin without cofactor), and leads to consecutive formation of sulfinate and sulfonate states of Cys69. Use of 7-chloro-4- nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reveals that Cys69 modification to a sulfenic acid is a transient intermediate during oxidation. Dithiothreitol converts sulfenic acid and disulfide into thiols, whereas the sulfinate and sulfonate forms of Cys69 are irreversible with respect to this reagent. A variable fraction of Cys69 in freshly isolated flavodoxin is in the sulfenic acid state, but neither oxidation to sulfinic and sul