High dissociation rate constant of ferrous-dioxy complex linked to the catalase-like activity in lactoperoxidase

Heme reduction of ferric lactoperoxidase (LPO) into its ferrous form initially leads to the accumulation of the unstable form of LPO-Fe(II), which spontaneously converts to a more stable species, the two of which can be identified by Soret peaks at 440 and 434 nm, respectively. Our data demonstrate that both LPO-Fe(II) species are capable of binding O₂ at a similar rate to generate the ferrous-dioxy complex. Its formation with respect to O ₂ was first order and monophasic and with rate constants k _on = 3.8 × 10⁴ M^-1 S^-1 and k_off = 11.2 s^-1. The dissociation rate constant for the formation of LPO-Fe(II)-O₂ is relatively high, in contrast to hemoprotein model compounds. This high dissociation rate can be attributed to a combination of effects that include the positive trans effect of the proximal ligand, the heme pocket environment, and the geometry of the Fe-O₂ linkage. Our results have also shown that the decay of the LPO-Fe(II)-O ₂ complex occurs by two sequential O₂-independent steps. The first step involves formation of a short-lived intermediate that can be characterized by its Soret absorption peak at 416 nm and may be attributed to the weakening of the Fe(II)-O₂ linkage with a rate constant of 0.5 s^-1. The second step is spontaneous conversion of this intermediate to generate the native enzyme and presumably superoxide as end products with a rate constant of 0.03 s^-1. A comprehensive kinetic model that links LPO-Fe(II)-O₂ complex formation to the LPO catalase-like activity, combined with the classic catalytic cycle, is presented here.