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 sulfonic acid nor formation of intermolecular disulfides is observed under oxidising conditions. Furthermore, flavodoxin does not react appreciably with NBD-Cl. Besides its primary role as redox-active moiety, binding of flavin leads to considerably improved stability against protein unfolding and to strong protection against irreversible oxidation and other covalent thiol modifications. Thus, cofactors can protect proteins against oxidation and modification.
Highlights
Proteins are sensitive to oxidative damage and the resulting protein modifications often have considerable biological effects
We propose that the species eluting at 510 mM KCl at pH 8 (Figure 3a), is protein with Cys69 in the sulfenic acid state (Figure 1), because it is converted into protein that elutes at 470 mM KCl after incubation with DTT (Figure 3b)
Because the pKa of sulfenic acid is about 5.9 [16], at pH 8 one expects retarded elution on an anion exchange column of the sulfenic acid state of flavodoxin compared to non-oxidised protein, just as we observe (Figure 3a)
Summary
Proteins are sensitive to oxidative damage and the resulting protein modifications often have considerable biological effects. Of particular interest is oxidation of methionine and cysteine residues, because this conversion happens for a wide variety of proteins and often affects their biological activity [4,5]. Oxidation of methionine to its sulfoxide state can be stimulated by biological oxidants such as H2O2, and by environmental oxidants like ozone [6]. Methionine oxidation can be (partially) reversed by the action of methionine sulfoxide reductases [7]. Such repair of oxidative damage suggests a potential for in vivo regulation of protein function by reversible methionine oxidation [8,9,10,11]
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