PMID: 2501303Jul 25, 1989Paper

The non-flavin redox center of the streptococcal NADH peroxidase. II. Evidence for a stabilized cysteine-sulfenic acid.

The Journal of Biological Chemistry
Leslie B Poole, A Claiborne

Abstract

Incubation of the streptococcal NADH peroxidase with 5-thio-2-nitrobenzoate under anaerobic denaturing conditions leads to the rapid incorporation of 1 eq/FAD of the aromatic thiol. Addition of dithiothreitol to the resulting conjugate, following ultrafiltration, demonstrates that a mixed disulfide has been formed. Analysis of the denatured NADH peroxidase by iso-electric focusing reveals the presence of two predominant species differing in isoelectric point by approximately 0.1 units. Preincubation with 20 mM hydrogen peroxide gives essentially complete and irreversible conversion to the more acidic species. Treatment of the native peroxidase with low concentrations of hydrogen peroxide also leads to irreversible enzyme inactivation; the low extinction long wavelength absorbance associated with the enzyme as purified is lost in the process. Anaerobic dithionite and NADH titrations of the peroxide-inactivated enzyme indicate that, while the cysteinyl redox center is nonfunctional, the enzyme is still capable of forming a binary complex with NADH. We propose that the redox-active cysteinyl derivative which serves as the second redox center in the native peroxidase is a stabilized cysteine-sulfenic acid derivative of Cys42. This ...Continue Reading

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