The non-flavin redox center of the streptococcal NADH peroxidase. II. Evidence for a stabilized cysteine-sulfenic acid.
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 determination is consistent with the covalent modifications observed with both 5-thio-2-nitrobenzoate and with H2O2 and is supported by mass spectrometric analysis of a chymotryptic cysteinyl peptide derived from the unmodified peroxidase.
Poole
L B
LB
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
Claiborne
A
A
D000595:Amino Acid Sequence; D003545:Cysteine / Q000031:analogs & derivatives*; D004229:Dithiothreitol / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D018377:Neurotransmitter Agents; D009579:Nitrobenzoates / Q000494:pharmacology; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D010544:Peroxidases / Q000378:metabolism*; D011489:Protein Denaturation; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds; D014357:Trypsin
D018377:Neurotransmitter Agents; D009579:Nitrobenzoates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C011136:thionitrobenzoic acid; D006861:Hydrogen Peroxide; D010544:Peroxidases; C021845:NAD+ peroxidase; D014357:Trypsin; D003545:Cysteine; D004229:Dithiothreitol; C013461:cysteine sulfinic acid