Iron Oxidation State Modulates Active Site Structure in a Heme Peroxidase † , ‡ Sandip K. Badyal, § Clive L. Metcalfe, § Jaswir Basran, | Igor Efimov, § Peter C. E. Moody, | and Emma Lloyd Raven* ,§ Department of Chemistry, Henry Wellcome Building, UniVersity of Leicester, UniVersity Road, Leicester LE1 7RH, England, U.K., and Department of Biochemistry and Henry Wellcome Laboratories for Structural Biology, Henry Wellcome Building, UniVersity of Leicester, Lancaster Road, Leicester LE1 9HN, England, U.K. ReceiVed NoVember 27, 2007; ReVised Manuscript ReceiVed February 14, 2008 ABSTRACT: We have previously shown [Badyal, S. K., et al. (2006) J. Biol. Chem. 281, 24512–24520] that the distal histidine (His42) in the W41A variant of ascorbate peroxidase binds to the heme iron in the ferric form of the protein but that binding of the substrate triggers a conformational change in which His42 dissociates from the heme. In this work, we show that this conformational rearrangement also occurs upon reduction of the heme iron. Thus, we present X-ray crystallographic data to show that reduction of the heme leads to dissociation of His42 from the iron in the ferrous form of W41A; spectroscopic and ligand binding data support this observation. Structural evidence indicates that heme reduction occurs through formation of a reduced, bis-histidine-ligated species that subsequently decays by dissociation of His42 from the heme. Collectively, the data provide clear evidence that conformational movement within the same heme active site can be controlled by both ligand binding and metal oxidation state. These observations are consistent with emerging data on other, more complex regulatory and sensing heme proteins, and the data are discussed in the context of our developing views in this area. The iron-containing heme group is used widely in biology. Traditionally, heme-containing proteins have been catego- rized into the oxygen transport proteins (the globins), the electron transfer proteins (the cytochromes), and the catalytic heme-containing enzymes (e.g., the P450s, peroxidases, etc.). This categorization conveniently differentiated the noncata- lytic electron transfer proteins from the transport proteins and the catalytic enzymes, and it became clear that at least part of this differentiation arose from differences in heme coordination geometry. Hence, the electron transfer proteins contain six-coordinated heme groups, as a means of facilitat- ing rapid electron transfer, whereas the transport proteins and the catalytic enzymes are most often found as five- coordinate or weakly six-coordinate heme structures to allow ligand binding or catalysis at the sixth site. Over the past several years, a few examples emerged in the literature of heme proteins that did not fit this categorization. This was because these proteins actually switched their heme coordi- nation geometry through conformational rearrangements of the protein structure. Examples include cytochrome c (1), Chlamydomonas hemoglobin (2), cytochrome cd 1 (3), the diheme cytochrome c peroxidase (4), the heme chaperone protein CcmE (5), and leghemoglobin (6). The trigger for these conformational rearrangements appeared to be, vari- ously, pH, the oxidation state of the iron, and the binding of ligands, substrate, or other (noncatalytic) metal ions. Al- though these documented examples were significant in their own right, their collective significance was not immediately apparent. This was in part because a functional basis for the ligand switch could not be identified in all cases. Later, further examples of conformational rearrangements in other regulatory heme proteins linked to, for example, gas-sensing processes, signaling, and gene transcription were published (see ref 7 for a recent review). It was only then that it started to become clear that conformational rearrangements associ- ated with the heme group, its ligands and/or substrates, and its oxidation state might actually be used more widely as a means of regulation and/or sensing in biology. Currently, therefore, it appears that at least some heme protein architectures are intrinsically mobile, that this mobil- ity can be triggered by redox changes or ligand-substrate binding, and that this trigger is used, in certain cases, as a link to more complex downstream biological processes. What we do not yet know is whether conformational mobility is a more general characteristic of other heme protein structures and whether these triggering mechanisms are more generally accessible in other protein structures. In this context, we have recently reported (8) an example of a six-coordinate heme peroxidase (W41A variant of ascorbate peroxidase) which has bis-histidine coordination, like a cytochrome, but that is catalytically active because the distal histidine reversibly dissociates to form a five-coordinate heme in response to binding of hydrogen peroxide. In this work, we show that this conformational movement is also triggered by a change in oxidation state. The implications of these observations are † This work was supported by grants from the BBSRC (Grants BB/ C00602X/1, BB/C001184/1, and IIP0206/009, and a studentship to S.K.B.) and ESRF Proposal MX-635. ‡ PDB accession codes for the protein structures reported in this paper are as follows: 2VNZ (ferrous W41A), 2VO2 (low-dose W41A), and 2VNX (high-dose W41A). * To whom correspondence should be addressed. Telephone: +44 (0)116 2297047. Fax: +44 (0)116 2522789. E-mail: emma.raven@ le.ac.uk. § Department of Chemistry, University of Leicester. | Department of Biochemistry and Henry Wellcome Laboratories for Structural Biology, University of Leicester. Biochemistry 2008, 47, 4403–4409 4403 10.1021/bi702337n CCC: $40.75  2008 American Chemical Society Published on Web 03/20/2008