US EPA

6840446 

Journal Article 

super(13)C NMR analysis of the cysteine-sulfenic acid redox center of enterococcal NADH peroxidase 

Crane, EJ III; Vervoort, J; Claiborne, AS 

1997 

36 

28 

8611-8618 

In order to characterize the native Cys42-sulfenic acid redox center of the flavoprotein NADH peroxidase by NMR, an expression protocol has been developed which yields the [3- super(13)C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-sulfenic acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH sub(2)) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-sulfenic acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-sulfenic acid to the Cys42-sulfinic and/or sulfonic acid states, gives rise to a new maximum in the difference spectrum of E sub(inactive) minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2- super(13)C]His-labeled peroxidase HHAA mutant (His sub(10)His sub(23)Ala sub(87)Ala sub(258)); the spectral change over the pH range 5.8-7.8 is attributed to deprotonation of the surface-exposed His23. Furthermore, replacement of Arg303, which is hydrogen bonded to His10, has no effect on the super(13)C spectrum. These results provide direct evidence in support of the peroxidase Cys42-sulfenic acid/thiol redox cycle and add significantly to our structure-based understanding of protein-sulfenic acid stabilization and function. 

Enterococcus faecalis N.M.R. spectroscopy reduction