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J. Biol. Chem., Vol. 281, Issue 34, 24512-24520, August 25, 2006

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Conformational Mobility in the Active Site of a Heme Peroxidase^*

Sandip K. Badyal, M. Gordon Joyce, Katherine H. Sharp, Harriet E. Seward, Martin Mewies, Jaswir Basran, Isabel K. Macdonald, Peter C. E. Moody, and Emma Lloyd Raven¹

From the Department of Chemistry, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom and Department of Biochemistry and Henry Wellcome Laboratories for Structural Biology, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.

Received for publication, March 20, 2006 , and in revised form, June 6, 2006.

The atomic coordinates and structure factors (code 2GGN, 2GHC, 2GHD, 2GHE, 2GHH, 2GHK) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Project Grants 91/B19083 and B11469 [GenBank] and a studentship from the Biotechnology and Biological Sciences Research Council (to S. K. B.) and by a fellowship from The Leverhulme Trust (to E. L. R.). 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.

¹ To whom correspondence should be addressed. Tel.: 44-116-229-7047; Fax: 44-116-252-2789; E-mail: emma.raven{at}le.ac.uk.

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