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J. Biol. Chem., Vol. 279, Issue 14, 13297-13300, April 2, 2004

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A Distinctive Electrocatalytic Response from the Cytochrome c Peroxidase of Nitrosomonas europaea^*

Amy L. Bradley, Sarah E. Chobot, David M. Arciero, Alan B. Hooper, and Sean J. Elliott¶

From the Department of Chemistry, Boston University, Boston, Massachusetts 02215 and the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota 55108

Here the cytochrome c peroxidase (CcP) from Nitrosomonas europaea is examined using the technique of catalytic protein film voltammetry. Submonolayers of the bacterial diheme enzyme at a pyrolytic graphite edge electrode give catalytic, reductive signals in the presence of the substrate hydrogen peroxide. The resulting waveshapes indicate that CcP is bound non-covalently in a highly active configuration. The native enzyme has been shown to possess two heme groups of low and high potential (L and H, –260 and +450 mV versus hydrogen, respectively), and here we find that the catalytic waves of the N. europaea enzyme have a midpoint potential of >500 mV and a shape that corresponds to a 1-electron process. The signals increase in magnitude with hydrogen peroxide concentration, revealing Michaelis-Menten kinetics and K_m = 55 µM. The midpoint potentials shift with substrate concentration, indicating the electrochemically active species observed in our data corresponds to a catalytic species. The potentials also shift with respect to pH, and the pH dependence is interpreted in terms of a two pK_a model for proton binding. Together the data show that the electrochemistry of the N. europaea cytochrome c peroxidase is unlike other peroxidases studied to date, including other bacterial enzymes. This is discussed in terms of a catalytic model for the N. europaea enzyme and compared with other cytochrome c peroxidases.

Received for publication, January 16, 2004 , and in revised form, February 16, 2004.

^* This work was supported by Boston University Start-Up Funds (to S. J. E.). 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: Dept. of Chemistry, Boston University, 590 Commonwealth Ave., Boston, MA 02215. Tel.: 617-358-2816; Fax: 617-353-6646; E-mail: elliott{at}bu.edu.

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