title: 13C NMR analysis of the cysteine-sulfenic acid redox center of enterococcal NADH peroxidase.
abstract: 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 [3-13C]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 (EH2) 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 Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled peroxidase HHAA mutant (His10His23Ala87Ala258); 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 13C 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.
authors.affiliation: Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Crane
authors.forename: J
authors.initials: J
authors.lastname: Vervoort
authors.forename: A
authors.initials: A
authors.lastname: Claiborne
mesh_terms: D001665:Binding Sites; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013293:Enterococcus faecalis / Q000201:enzymology*; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry*; D010545:Peroxides / Q000494:pharmacology; D013434:Sulfenic Acids / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005420:Flavoproteins; D010545:Peroxides; D013434:Sulfenic Acids; D009243:NAD; D010544:Peroxidases; C021845:NAD+ peroxidase; C100870:cysteinesulfenic acid; D003545:Cysteine