Resonance Raman Spectroscopy Reveals pH-Dependent Active Site Structural Changes of Lactoperoxidase Compound 0 and Its Ferryl Heme O–O Bond Cleavage Products

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.

Unveiling the crucial intermediates in androgen production

“…Although the observation of this single feature, appearing at 791 cm −1 , is obviously consistent with an intermediate with "peroxo-like" character, it must be noted that this vibrational frequency and 16 O/ 18 O isotopic shifts are close to what is expected for the ν(Fe = O) mode of a ferryl heme species; the frequencies of such ferryl species depend on the transaxial ligand and distal pocket interactions, with reported values ranging from 745 cm −1 up to about 800 cm −1 (34,36). However, the assignment of this feature to a ferryl species was ruled out by experiments conducted with scrambled oxygen, a (1:2:1) mixture of 16 Fig.…”

“…Having confirmed the existence of a 405-nm intermediate for the sample containing 17-OH PREG, we exploited the impressive power of rR spectroscopy, which is able to provide definitive structural characterization of such trapped species, revealing telltale shifts of the internal vibrational modes of the Fe-O-O fragments in response to even quite subtle, but functionally significant, active site structural changes (23,34). The essential results of such studies are collected in Fig.…”

The catalytic mechanism of A-type dye-decolourising peroxidase BsDyP: neither aspartate nor arginine is individually essential for peroxidase activity

“…[29][30][31][32]35 We show that Cpd I formation in the reaction of BsDyP and H 2 O 2 is strongly pH dependent as well as its spontaneous decay to Cpd II. The kinetic analysis of the reaction of guaiacol with Cpd I and Cpd II demonstrates the formation of a pre-equilibrium binding of guaiacol to Cpd II prior to rate-limiting electron transfer, which constitutes the limiting step in the overall BsDyP catalytic cycle.…”

“…These include low temperature studies on horseradish peroxidase (HRP) [29][30][31][32] , HRP variants 33,34 and lactoperoxidase, using cryoradiolysis and RR spectroscopy. 35 The second order rate constant of Cpd I formation (k 1 ' = k 1 / K 1 ), ((1.8 ± 0.1) × 10 6 M -1 s -1 ) is of the same magnitude as those previously reported for the HRP, 34,36 lignin and manganese peroxidase from the white-rot fungus P. chrysosporium 37,38 and versatile peroxidase from Pleurotus eryngii 39 and it is one order of magnitude higher than that of bacterial DyPB from R. jostii RHA1. 22 Cpd I (A and B (a)) and Cpd II (B (b)) formation as a function of time were followed at 397 and 420 nm, respectively and the rate constants were obtained from single exponential fits (Fig.…”

Application of resonance Raman spectroscopy for interrogation of cryoradiolytically reduced oxygenated heme proteins

“…For many years, the resonance Raman (rR) technique has proven itself to be of immense value for probing not only stable terminal species, but also reaction intermediates, such as the ferryl species that arise from proton assisted rapid O―O bond cleavage processes, including Compounds I and II encountered in the catalytic cycles of peroxidases, cytochromes P450 and related enzymes . However, it has been only recently that rR spectroscopy has been successfully applied to interrogate the primary intermediates, that is the peroxo‐ and hydroperoxo‐ forms, by exploiting cryoradiolysis, which effectively restricts proton transfer, with most of the studies being focused on cytochromes P450 …”