Resonance Raman Spectroscopy Reveals pH-Dependent Active Site Structural Changes of Lactoperoxidase Compound 0 and Its Ferryl Heme O–O Bond Cleavage Products
Document Type
Article
Language
eng
Format of Original
13 p.
Publication Date
2015
Publisher
American Chemical Society
Source Publication
Journal of the American Chemical Society
Source ISSN
0002-7863
Original Item ID
DOI: 10.1021/ja5107833
Abstract
The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this “stored” proton to the hydroperoxo fragment, it rapidly undergoes O–O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe–O–O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O–O bond-cleavage product, whose ν(Fe═O) frequencies reflect substantially different Fe═O bond strengths. In the process of conducting these studies, rR structural characterization of the dioxygen adduct of LPO, commonly called Compound III, has also been completed, demonstrating a substantial difference in the strengths of the Fe–O linkage of the Fe–O–O fragment under acidic and alkaline conditions, an effect most reasonably attributed to a corresponding weakening of the trans-axial histidyl imidazole linkage at lower pH. Collectively, these new results provide important insight into the impact of pH on the disposition of the key Fe–O–O and Fe═O fragments of intermediates that arise in the enzymatic cycles of LPO, other mammalian peroxidases, and related proteins.
Recommended Citation
Mak, Piotr J.; Thammawichai, Warut; Wiedenhoeft, Dennis; and Kincaid, James R., "Resonance Raman Spectroscopy Reveals pH-Dependent Active Site Structural Changes of Lactoperoxidase Compound 0 and Its Ferryl Heme O–O Bond Cleavage Products" (2015). Chemistry Faculty Research and Publications. 520.
https://epublications.marquette.edu/chem_fac/520
Comments
Accepted version. Journal of the American Chemical Society, Vol. 137, No. 1 (2015): 349-361. DOI. © 2014 American Chemical Society. Used with permission.