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 oxyheme 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 α-meso-hydroxyhemin-HO product. In contrast, oneelectron 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.

The Influence of X‐Rays on the Structural Studies of Peroxide‐Derived Myoglobin Intermediates

“…This is typical for low-temperature oxyMb spectra [104]. It has been shown that the oxyMb is quite easily one-electron-reduced by 60 Co g-irradiation [67] [104], as well as oxyP450, oxyHRP, and oxyCPO [29] [105] [106]. Similar spectroscopic changes as observed by 60 Co g-irradiation are observed for synchrotron X-ray-irradiated Mb compound III (Fig.…”

“…An issue to be raised is if it is the peroxyMb or the hydroperoxyMb state that has been trapped and solved with X-ray diffraction. Optical absorption, EPR, and Mçssbauer spectroscopy have shown that the cryoradiolytically reduced oxyMb is a peroxyMb state, and that temperatures near 185 K are needed to induce a proton transfer resulting in hydroperoxyMb [28] [67]. The ca.…”

“…-Cryoradiolytical reduction of oxyMb generates peroxyMb, and it has been shown by EPR spectroscopy that an annealing to temperatures 190 -200 K generates the hydroperoxyMb [128 -130]. Mçssbauer studies with annealing to 195 K have, however, resulted in the breaking of the OÀO bond forming Mb compound II [67]. This is different from oxyHO that is cryoradiolytically reduced to hydroperoxyHO at 77 K (also with amounts of ferrylHO), which, by annealing, propagates to the a-mesohydroxyhemin HO product [67].…”

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

“…These fleeting intermediates of heme enzyme catalysis could not be isolated in ambient conditions, despite numerous attempts. However, being immersed in liquid nitrogen, they are stable for many months and can be studied using various spectroscopic methods, such as EPR (62,65,67,73-76), UV-Vis absorption spectroscopy (58,59,69,70,76-79), resonance Raman (69,71,72,79,80), Mössbauer (67), and EXAFS (81). …”

Electronic structure, chemical bonding, and oxidation numbers of first‐row transition metals in [MePIm2] complexes and their cations

“…It can also be noted that the conclusion that the HOMO of heme group is formed by d orbitals of Fe atom rather than orbitals of porphyrin ring has been derived from interpretation of the changes in the Mössbauer spectra of cytochromes before and after redox reactions [19][20][21] To reduce computational complexity of the quantum mechanical calculations, some simplifications have been used. For this reason, the present investigation has been extended to similar complexes of all first-row transition metals.…”