title: 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.
abstract: The trapping of a sulfenic acid within the fully active C165S mutant of the AhpC peroxidase protein from Salmonella typhimurium was investigated. The electrophilic reagent employed in these studies, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has previously been used to modify 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 with an absorbance maximum at 347 nm, whereas the thiol-NBD conjugate formed from the reduced protein absorbed maximally at 420 nm. Electrospray ionization mass spectrometry of the modified proteins allowed identification of the species absorbing at 347 nm as a Cys-S(O)-NBD derivative 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 peroxidase activity when compared to unreacted C165S and wild-type AhpC, but were both reactivated through removal of NBD by DTT. Oxidized C165S was also modified by dimedone, a common sulfenic acid reagent, to give the expected inactivated conjugate of higher mass. This reagent was not removed by DTT and blocked any further reaction of the protein with NBD-Cl. NBD modification of Enterococcus faecalis NADH peroxidase, a well-characterized flavoprotein with an active-site sulfenic acid (Cys-SOH), also yielded the spectrally-distinguishable NBD conjugates following incubation of NBD-Cl with oxidized and reduced forms of the denatured peroxidase, indicating a general utility for this reagent with other sulfenic acid-containing proteins. A significant advantage of NBD-Cl over previously-used sulfenic acid reagents such as dimedone is in the retention of the sulfenic acid oxygen in the modified product; differentiation between protein-associated thiols and sulfenic acids is therefore now possible by means of both visible absorbance properties and mass analyses of the NBD-modified proteins.
authors.affiliation: Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
authors.forename: H R
authors.initials: HR
authors.lastname: Ellis
authors.forename: L B
authors.initials: LB
authors.lastname: Poole
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D003512:Cyclohexanones / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D013058:Mass Spectrometry; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054464:Peroxiredoxins; D012486:Salmonella typhimurium / Q000201:enzymology*; D013053:Spectrophotometry; D013434:Sulfenic Acids / Q000302:isolation & purification*; D013438:Sulfhydryl Compounds / Q000302:isolation & purification
chemical_list: D003512:Cyclohexanones; D013434:Sulfenic Acids; D013438:Sulfhydryl Compounds; C004774:dimedone; D010088:Oxidoreductases; D010544:Peroxidases; C021845:NAD+ peroxidase; D054464:Peroxiredoxins; D009327:4-Chloro-7-nitrobenzofurazan; C100870:cysteinesulfenic acid; D003545:Cysteine