NOVEL APPLICATION OF 7-CHLORO-4-NITROBENZO-2-OXA-1,3-DIAZOLE TO IDENTIFY CYSTEINE SULFENIC ACID IN THE AHPC COMPONENT OF ALKYL HYDROPEROXIDE REDUCTASE

The trapping of a sulfenic acid within the fully active C165S mutant o f the AhpC peroxidase protein from Salmonella typhimurium was investig ated. The electrophilic reagent employed in these studies, 7-chloro-4- nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has previously been used to modi fy thiol, amino, and tyrosine hydroxyl groups in proteins; at neutral pH only cysteinyl residues of AhpC proteins are modified. The peroxide -oxidized C165S mutant of AhpC incubated with NBD-Cl gave a product wi th an absorbance maximum at 347 nm, whereas the thiol-NBD conjugate fo rmed from the reduced protein absorbed maximally at 420 nm. Electrospr ay ionization mass spectrometry of the modified proteins allowed ident ification of the species absorbing at 347 nm as a Cys-S(O)-NBD derivat ive containing one additional oxygen relative to the Cys-S-NBD product . The C165S conjugates with Cys-S(O)-NBD and Cys-S-NBD had no peroxida se activity when compared to unreacted C165S and wild-type AhpC, but w ere both reactivated through removal of NBD by DTT. Oxidized C165S was also modified by dimedone, a common sulfenic acid reagent, to give th e expected inactivated conjugate of higher mass. This reagent was not removed by DTT and blocked any further reaction of the protein with NB D-Cl. NBD modification of Enterococcus faecalis NADH peroxidase, a wel l-characterized flavoprotein with an active-site sulfenic acid (Cys-SO H), also yielded the spectrally-distinguishable NBD conjugates followi ng incubation of NBD-Cl with oxidized and reduced forms of the denatur ed peroxidase, indicating a general utility for this reagent with othe r sulfenic acid-containing proteins. A significant advantage of NBD-C1 over previously-used sulfenic acid reagents such as dimedone is in th e retention of the sulfenic acid oxygen in the modified product; diffe rentiation between protein-associated thiols and sulfenic acids is the refore now possible by means of both visible absorbance properties and mass analyses of the NBD-modified proteins.