The non-flavin redox center of the streptococcal NADH peroxidase. I. Thiol reactivity and redox behavior in the presence of urea.
Unlike the 2-electron-reduced (EH2) forms of the flavoprotein disulfide reductases and mercuric reductase, the native EH2 form of the streptococcal NADH peroxidase is quite refractile toward chemical modification with thiol-specific reagents. In the presence of 1.3 M urea, however, the single thiol of the reduced enzyme reacts with phenylmercuric acetate with a t1/2 of 3 min. This modification abolishes the charge-transfer absorbance band at 540 nm and inactivates the enzyme; the latter effect is shown to be reversed with dithiothreitol. Alkylation of the streptococcal peroxidase with iodo[1-14C]acetamide under reducing conditions in the presence of 8 M guanidine hydrochloride allows the isolation of a single labeled tryptic peptide with the sequence: Gly-Asp-Phe-Ile-Ser-Phe-Leu-Ser-C*ys-Gly-Met-Gln-Leu-Tyr-Leu- Glu-Gly-Lys. This sequence is identical to that previously reported (Poole, L. B., and Claiborne, A. (1988) Biochem. Biophys. Res. Commun. 153, 261-266) for the cysteinyl peptide isolated from the NADH peroxidase labeled metabolically with [35S]cysteine. Careful examination of the physical properties of the streptococcal peroxidase in the presence of 1.3 M urea shows that, while catalytic activity and native structural features are largely retained, the relative potentials of flavin and non-flavin redox centers are dramatically affected. We propose that low concentrations of urea stabilize an intermediate state in the transition between native and denatured forms, which is responsible for the observed changes in both active-site thiol reactivity and in redox properties.
Poole
L B
LB
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
Claiborne
A
A
D000595:Amino Acid Sequence; D013293:Enterococcus faecalis / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D007460:Iodoacetamide / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D014357:Trypsin; D014508:Urea / Q000494:pharmacology*
D010446:Peptide Fragments; D014508:Urea; D010544:Peroxidases; C021845:NAD+ peroxidase; D005979:Glutathione Peroxidase; D014357:Trypsin; D007460:Iodoacetamide