The non-flavin redox center of the streptococcal NADH peroxidase. II. Evidence for a stabilized cysteine-sulfenic acid.
作者： L B Poole , A Claiborne
DOI: 10.1016/S0021-9258(18)63862-1
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摘要: Abstract Incubation of the streptococcal NADH peroxidase with 5-thio-2-nitrobenzoate under anaerobic denaturing conditions leads to rapid incorporation 1 eq/FAD aromatic thiol. Addition dithiothreitol resulting conjugate, following ultrafiltration, demonstrates that a mixed disulfide has been formed. Analysis denatured by iso-electric focusing reveals presence two predominant species differing in isoelectric point approximately 0.1 units. Preincubation 20 mM hydrogen peroxide gives essentially complete and irreversible conversion more acidic species. Treatment native low concentrations also enzyme inactivation; extinction long wavelength absorbance associated as purified is lost process. Anaerobic dithionite titrations peroxide-inactivated indicate that, while cysteinyl redox center nonfunctional, still capable forming binary complex NADH. We propose redox-active derivative which serves second stabilized cysteine-sulfenic acid Cys42. This determination consistent covalent modifications observed both H2O2 supported mass spectrometric analysis chymotryptic peptide derived from unmodified peroxidase.
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