PROBING THE HEME IRON COORDINATION STRUCTURE OF ALKALINE CHLOROPEROXIDASE

The mechanism by which the heme-containing peroxidase, chloroperoxidas e, is able to chlorinate substrates is poorly understood. One approach to advance our understanding of the mechanism of the enzyme is to det ermine 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 ch loroperoxidase (C420) have been characterized by electronic absorption , magnetic circular dichroism, and electron paramagnetic resonance spe ctroscopy. The heme iron oxidation state influences the transition to C420; the pK(a) for the alkaline transition is 7.5 for the ferric prot ein and 9.5 for the ferrous protein, The five-coordinate, high-spin fe rric native protein converts to a six-coordinate low-spin species (C42 0) as the pH is raised above 7.5. The inability of ferric C420 to bind exogenous ligands, as well as the dramatically increased reactivity o f 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 peroxi de binding site while increasing the accessibility of Cys29. However, it does appear that Cys29-derived ligation is at least partially retai ned by ferric C420, potentially in a thiolate/imidazole coordination s phere. Ferrous C420, on the other hand, appears not to possess a thiol ate ligand but instead likely has a bis-imidazole (histidine) coordina tion structure. The axial ligand trans to carbon monoxide in ferrous-C O C420 may be a histidine imidazole. Since chloroperoxidase functions normally through the ferric and higher oxidation states, the fact that the proximal thiolate ligand is largely retained in ferric C420 clear ly indicates that additional factors such as the absence of a vacant s ixth coordination site sufficiently accessible for peroxide binding ma y be the cause of catalytic inactivity.