A nuclear paramagnetic relaxation study of the interaction of the cyclopentanedione substrate with chloroperoxidase

Association of the physiological substrate cyclopentanedione (CPDO) with chloroperoxidase (CPO) was confirmed from both line-width and longitudinal relaxation time measurements of the proton NMR signals of CPDO. A dissociation constant of 33 mM for the enzyme-substrate complex was calculated from proton NMR relaxation experiments. Chloride ion does not affect the stability of the complex, but iodide ion greatly decreased the stability of the CPO-CPDO complex. Binding of cyanide ion to the heme iron center of CPO also decreased the stability of the CPO-CPDO complex. This result indicates the critical influence of perturbation in the heme active site on the affinity of CPO toward its substrate. Titration of CPO with CPDO revealed that CPDO does not bind directly to the heme iron of CPO. A distance of 7.1 A between the heme center of CPO and the protons of bound CPDO is deduced from the Solomon-Bloembergen relationship. This distance is 1 to 3 A shorter than those reported for the complexes formed between CPO and organic sulfide and unreactive phenol substrates, but the distance approximates that for reactive phenol substrates.