Distinct reaction pathways followed upon reduction of oxy-heme oxygenase and oxy-myoglobin as characterized by Mössbauer spectroscopy

Abstract

Activation of O(2) by heme-containing monooxygenases generally commences with the common initial steps of reduction to the ferrous heme and binding of O(2) followed by a one-electron reduction of the O(2)-bound heme. Subsequent steps that generate reactive oxygen intermediates diverge and reflect the effects of protein control on the reaction pathway. In this study, Mössbauer and EPR spectroscopies were used to characterize the electronic states and reaction pathways of reactive oxygen intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of oxy-heme oxygenase (HO) and oxy-myoglobin (Mb). The results confirm that one-electron reduction of (Fe(II)-O(2))HO is accompanied by protonation of the bound O(2) to generate a low-spin (Fe(III)-O(2)H(-))HO that undergoes self-hydroxylation to form the alpha-meso-hydroxyhemin-HO product. In contrast, one-electron reduction of (Fe(II)-O(2))Mb yields a low-spin (Fe(III)-O(2)(2-))Mb. Protonation of this intermediate generates (Fe(III)-O(2)H(-))Mb, which then decays to a ferryl complex, (Fe(IV)=O(2-))Mb, that exhibits magnetic properties characteristic of the compound II species generated in the reactions of peroxide with heme peroxidases and with Mb. Generation of reactive high-valent states with ferryl species via hydroperoxo intermediates is believed to be the key oxygen-activation steps involved in the catalytic cycles of P450-type monooxygenases. The Mössbauer data presented here provide direct spectroscopic evidence supporting the idea that ferric-hydroperoxo hemes are indeed the precursors of the reactive ferryl intermediates. The fact that a ferryl intermediate does not accumulate in HO underscores the determining role played by protein structure in controlling the reactivity of reaction intermediates.

Figure 1

Mössbauer spectra of the as-prepared oxygenated HO sample before (A and B) and after (C) radiolytic reduction at 77K. The spectra (hatched marks) were recorded at 4.2 K in a magnetic field of 8 T (A), or 50 mT (B and C) applied parallel to the γ beam. The theoretical spectra of the individual species are shown as color lines above the experimental spectra ((Fe^II-O₂)HO, red; (Fe^III-H₂O)HO, cyan; (Fe^III-O₂H⁻)HO, blue; low-spin Fe^II HO, purple; (Fe^IV=O²⁻)HO, green), and the composite spectra are plotted as black solid lines overlaid with the experimental data.

Figure 2

Mössbauer spectra of (Fe^III-O₂H⁻)HO prepared from the raw spectra recorded at 4.2 K in a parallel field of 8 T (A), 4 T (B) or 50 mT (C), and in a perpendicular field of 50 mT (D) (see text for details). The blue solid lines are theoretical spectra simulated with the parameters listed in Table 2.

Figure 3

Mössbauer spectra of the cryoreduced oxygenated HO sample after annealing at 220 K. The data (hatched marks) were recorded at 4.2 K in a parallel applied field of 50 mT (A) or 4 T (B). The theoretical spectra of each individual species are shown as color lines above the experimental spectra ((Fe^II-O₂)HO, red; Fe^III-α-hydroxy-HO plus (Fe^III-H₂O)HO, cyan; (Fe^III-O₂H⁻)HO, blue; low-spin Fe^II, purple; (Fe^IV=O²⁻)HO, green; high-spin Fe^II HO, orange), and the composite spectra are shown as black solid line overlaid with the experimental spectra.

Figure 4

Mössbauer spectra of the as-prepared oxygenated Mb sample before (A and B) and after (C) radiolytic reduction at 77K. The spectra (hatched marks) were recorded at 4.2 K in a parallel field of 8 T (A) or 50 mT (B and C). The theoretical spectra of the individual species are shown as color lines above the experimental spectra ((Fe^II- O₂)Mb, red; (Fe^III-H₂O)Mb, cyan; (Fe^III-O₂²⁻)Mb, blue; low-spin Fe^II Mb, purple), and the composite spectra are plotted as black solid lines overlaid with the experimental data.

Figure 5

EPR spectra of cryoreduced oxy Mb (A). (B) and (C) are spectra of this same sample after annealing at 180 K and 190 K, respectively. The spectra are recorded at 77 K, 9.10 GHz, 10 mW microwave power, 100 kHz modulation frequency and 0.5 mT modulation amplitude.

Figure 6

Mössbauer spectra of (Fe^III-O₂²⁻)Mb prepared from the raw spectra recorded at 4.2 K in a parallel field of 8 T (A), 4 T (B) or 50 mT (C) (see text for details). The blue solid lines are theoretical spectra simulated with the parameters listed in Table 2.

Figure 7

Mössbauer spectra of the cryoreduced oxygenated HO sample after annealing at 180 K (A) and at 195 K (B). The data (hatched marks) were recorded at 4.2 K in a parallel applied field of 50 mT. The theoretical spectra of each individual species are shown as color lines above the experimental spectra ((Fe^II-O₂)Mb, red; high-spin Fe^III Mb, cyan in A; (Fe^III-O₂ ²⁻)Mb, blue; low-spin Fe^II Mb, purple; (Fe^IV=O²⁻)Mb, green; deoxy Fe^II Mb, orange; the cyan line in B is the sum of (Fe^III-H₂O)Mb and Fe^III Mb product.), and the composite spectra are shown as black solid lines overlaid with the experimental spectra. A difference spectrum of B minus A is shown in C (hatched marks). The black solid line in C is the theoretical difference spectrum of (Fe^IV=O²⁻)Mb plus Fe^III Mb product minus (Fe^III-O₂²⁻)Mb.

Figure 8

Mössbauer spectra of (Fe^IV=O²⁻)Mb prepared from the raw spectra recorded at 4.2 K in a parallel field of 8 T (A) or 50 mT (C) (see text for details). The green solid lines are theoretical spectra simulated with the parameters listed in Table 2.