Identification of cysteine sulfenic acid in AhpC of alkyl hydroperoxide reductase.

摘要: Summary C165S AhpC in its sulfenate (Cys-SO−) and presumed thiolate (Cys-S−) forms at pH 7 (pKa for sulfenic acid about 6.1) exhibit low extinction absorbance bands around 367 324 nm, respectively. Sulfenic content of the protein can be assessed by reactivity with chromophoric TNB anion. Using this technique, H2O2 titrations give a maximum 1 SOH per subunit on addition 1.0 to 1.2 equivalents H2O2. Cys46-SO− is moderately air stable neutral room temperature oxidized steady rate 10% half hour. reduced presence catalytic amounts AhpF ∼1 equivalent NADH regenerate Cys46-S− species. NBD chloride extremely useful as trapping agent cysteine acid. The Cys46-S(O)-NBD adduct absorbs maximally 347 nm 16 amu larger than Cys46-S-NBD (γmax = 420 nm) shown ESI-MS. Other electrophilic thiol reagents also react Cys46-SO−; however, iodoacetamide N-ethylmaleimide reactivities are much lower Cys46-S−. These methods applicable other acid-containing proteins, although some cases proteins must denatured order provide accessibility species toward labeling agents.