Moessbauer and electron paramagnetic resonance studies of horseradish peroxidase and its catalytic intermediates

Abstract

We report Mössbauer and EPR measurements on horseradish peroxidase in the native state and the reaction intermediates with peroxide and chlorite. A detailed analysis of the electronic state of the heme iron is given, and comparisons are drawn with related systems. The native enzyme is high-spin ferric and thus has three Kramers doublets. The unusual magnetic properties of the ground doublet and the large energy of the second, (E2-E1)/k approximately equal to 41 K, and third doublet, (E3-E1)/k greater than or equal to 170 K, can be modeled with a quartet admixture of approximately 11% to the spin sextet. All evidence suggests a ferryl, OFeIV, state of the heme iron in compounds I and II and related complexes. The small isomer shift, delta Fe approximately equal to 0.06 mm/s, the (positive) quadrupole splitting, delta EQ approximately equal to 1.4 mm/s, the spin S = 1, and the large positive zero field splitting, D/k approximately equal to 35 K, are all characteristic of the ferryl state. In the green compound I the iron weakly couples to a porphyrin radical with spin S' = 1/2. A phenomenological model with a weak exchange interaction S . J . S', magnitude of less than or equal to 0.1 D, reproduces all Mössbauer and EPR data of compound I, but the structural origin of the exchange and its apparent distribution require further study. Reaction of horseradish peroxidase with chlorite leads to compound X with delta Fe = 0.07 mm/s and delta EQ = 1.53 mm/s, values that are closest to those of compound II. The diamagnetism of compound III and its Mössbauer parameters delta Fe = 0.23 mm/s and delta EQ = -2.31 mm/s at 4.2 K clearly identify it as an oxyheme adduct.

Detection of a Tryptophan Radical as an Intermediate Species in the Reaction of Horseradish Peroxidase Mutant (Phe-221 → Trp) and Hydrogen Peroxide

“…In contrast to that of the parent enzyme, the F221W mutant exhibited a rather sharp EPR signal at g ʈ ϭ 2.036 and g Ќ ϭ 2.007 (Fig. 3, b and c), characteristic of the Trp radical in CcP compound I (g ʈ ϭ 2.037 and g Ќ ϭ 2.005) (23)(24)(25). Although we have not yet succeeded in the quantitative analysis for the EPR measurements after mixing the mutant with hydrogen peroxide, the second reaction intermediate in which the Soret peak appeared at 419 nm seems to afford these EPR signals characteristic of the Trp radical.…”

Monoamine-dependent Production of Reactive Oxygen Species Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin

“…21,22) This intermediate is formed by accepting both of the oxidizing equivalents of H2O2, thus Compound I contains 2 additional oxidizing equivalents over the native ferric form of the enzyme. 21,23) The heme iron of Compound I is known to be in the ferryl (Fe IV ＝O) state, 24) but the absorption maxima of Compounds I of various proteins have varied features. This has been attributed to the localization site of the second oxidizing equivalent.…”

“…This has been attributed to the localization site of the second oxidizing equivalent. 23) For HRP, 24) chloroperoxidase, 25) plant ascorbate peroxidase, 26) prostaglandin H synthase, 27) and lignin peroxidase, 23) the second oxidizing equivalent is stored as a porphyrin p-cation radical. However, for compound ESs of yeast and Pseudomonas cytochrome c peroxidase, 28,29) horse myoglobin, 30) leghemoglobin (Lb), 31) and human Hb, 7) the second oxidizing equivalent is on an amino acid side chain of the protein.…”

The biology and chemistry of high-valent iron–oxo and iron–nitrido complexes

“…Whereas δ and ∆E Q values (Table 1) obtained from zero-field Mössbauer studies of the active oxidants in both haem and non-haem oxygenases are consistent with an iron(IV) oxidation state, the analysis of the Mössbauer spectra in an applied magnetic field reveals different spin states in the two cases. For haem enzymes the A tensor shows a qualitative trend of 'two large negative values, one small negative value' 20,21 , thereby reflecting an intermediate spin, S = 1 spin state for the iron(IV) centre. In CPd-I intermediates, this S = 1 spin state is coupled ferromagnetically or antiferromagnetically to the porphyrin radical cation, giving an overall quartet or doublet state, respectively 3,6,7,9 .…”