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

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