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

• Published in: Biochemistry (Easton) Vol. 36; no. 48; pp. 15013 - 15018
• Main Authors: Ellis, Holly R, Poole, Leslie B
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.12.1997
• Subjects: 4-Chloro-7-nitrobenzofurazan - chemistry; Cyclohexanones - chemistry; Cysteine - analogs & derivatives; Cysteine - isolation & purification; Mass Spectrometry; Mutation; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Peroxidases - chemistry; Peroxidases - genetics; Peroxidases - metabolism; Peroxiredoxins; Salmonella typhimurium - enzymology; Spectrophotometry; Sulfenic Acids - isolation & purification; Sulfhydryl Compounds - isolation & purification
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi972191x

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.