The generation of a hyperporphyrin spectrum upon thiol binding to ferric chloroperoxidase. Further evidence of endogenous thiolate ligation to the ferric enzyme.
In spite of numerous spectroscopic similarities between chloroperoxidase and cytochrome P-450 (P-450) which suggest endogenous cysteinate axial ligation in chloroperoxidase as has been established for P-450, assignment of the endogenous axial ligand of chloroperoxidase has remained controversial since no available free sulfhydryl groups have been detected in chemical studies of chloroperoxidase. To help clarify this problem, we have carried out extensive studies of thiol-binding properties of native ferric chloroperoxidase and have compared our new results with those previously obtained in our laboratory on thiol adducts of P-450. We have found that the ligation of exogenous thiols to the heme iron of chloroperoxidase generates hyperporphyrin (split Soret) spectra (lambda max = approximately 372 and approximately 455 nm), consistent with the formation of bisthiolate low-spin ferric heme adducts as has been established for P-450 and its heme models. However, in contrast to the results with P-450, thiols not only coordinate to the ferric heme iron of chloroperoxidase in the thiolate form in competition with cyanide but also bind, presumably in the thiol form, to a site in the heme vicinity other than the heme iron without competition with cyanide. The thiol acidity (pK alpha greater than 7) or medium pH (pH 3-7) have little effect on the spectral and equilibrium properties of the adducts. Thiol binding to the latter site, which is presumably the organic substrate-binding site, causes a red shift of the Soret peak (339 vector approximately 420 nm) of the high-spin ferric enzyme; the resulting thiol adducts are still predominantly high spin. Similar spectral changes are also observed upon binding of other neutral sulfur (sulfides and disulfide) and oxygen (alcohols and ketones) donor ligands to native ferric chloroperoxidase. In conclusion, the generation of hyperporphyrin spectra upon exogenous thiol binding to native ferric chloroperoxidase provides considerable support for the presence of an endogenous thiolate ligand to the heme of the enzyme in its ferric state.
D000438:Alcohols / Q000378:metabolism; D001665:Binding Sites; D002711:Chloride Peroxidase / Q000378:metabolism*; D003545:Cysteine; D008024:Ligands; D003904:Mitosporic Fungi / Q000201:enzymology; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D011166:Porphyrins / Q000032:analysis*; D011485:Protein Binding; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D000438:Alcohols; D008024:Ligands; D011166:Porphyrins; D013438:Sulfhydryl Compounds; D010544:Peroxidases; D002711:Chloride Peroxidase; D003545:Cysteine