Abstract

The mechanism by which the heme-containing peroxidase, chloroperoxidase, is able to chlorinate substrates is poorly understood. One approach to advance our understanding of the mechanism of the enzyme is to determine those factors which contribute to its stability. In particular, under alkaline conditions, chloroperoxidase undergoes a transition to a new, spectrally distinct form, with accompanying loss of enzymatic activity. In the present investigation, ferric and ferrous alkaline chloroperoxidase (C420) have been characterized by electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy. The heme iron oxidation state influences the transition to C420; the pK(a) for the alkaline transition is 7.5 for the ferric protein and 9.5 for the ferrous protein. The five-coordinate, high-spin ferric native protein converts to a six-coordinate low-spin species (C420) as the pH is raised above 7.5. The inability of ferric C420 to bind exogenous ligands, as well as the dramatically increased reactivity of the proximal Cys29 heme ligand toward modification by the sulfhydryl reagent p-mercuribenzoate, suggests that a conformational change has occurred during conversion to C420 that restricts access to the peroxide binding site while increasing the accessibility of Cys29. However, it does appear that Cys29-derived ligation is at least partially retained by ferric C420, potentially in a thiolate/imidazole coordination sphere. Ferrous C420, on the other hand, appears not to possess a thiolate ligand but instead likely has a bis-imidazole (histidine) coordination structure. The axial ligand trans to carbon monoxide in ferrous-CO C420 may be a histidine imidazole. Since chloroperoxidase functions normally through the ferric and higher oxidation status, the fact that the proximal thiolate ligand is largely retained in ferric C420 clearly indicates that additional factors such as the absence of a vacant sixth coordination site sufficiently accessible for peroxide binding may be the cause of catalytic inactivity.

Original languageEnglish (US)
Pages (from-to)14537-14543
Number of pages7
JournalBiochemistry
Volume35
Issue number46
DOIs
StatePublished - Nov 19 1996

ASJC Scopus subject areas

  • Biochemistry

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