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J. Biol. Chem., Vol. 279, Issue 38, 39465-39470, September 17, 2004

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High Dissociation Rate Constant of Ferrous-Dioxy Complex Linked to the Catalase-like Activity in Lactoperoxidase^*

Semira Galijasevic, Ghassan M. Saed, Michael P. Diamond, and Husam M. Abu-Soud¶

From the Departments of Obstetrics and Gynecology and Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201

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 O₂ was first order and monophasic and with rate constants of k_on = 3.8 x 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)-O₂ 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.

Received for publication, May 28, 2004 , and in revised form, July 16, 2004.

^* This work was supported by National Institutes of Health Grant HL066367 (to H. M. A.-S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Wayne State University School of Medicine, Dept. of Obstetrics and Gynecology, The C. S. Mott Center for Human Growth and Development, 275 E. Hancock, Detroit, MI 48201. Tel.: 313-577-6178; Fax: 313-577-8554; E-mail: habusoud{at}med.wayne.edu.

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