Spectroscopic evidence for the presence of a high-valent Fe(IV) species in the ferroxidase reaction of an archaeal ferritin

Abstract

A high-valent Fe(IV) species is proposed to be generated from the decay of a peroxodiferric intermediate in the catalytic cycle at the di-iron cofactor center of dioxygen-activating enzymes such as methane monooxygenase. However, it is believed that this intermediate is not formed in the di-iron substrate site of ferritin, where oxidation of Fe(II) substrate to Fe(III) (the ferroxidase reaction) occurs also via a peroxodiferric intermediate. In opposition to this generally accepted view, here we present evidence for the occurrence of a high-valent Fe(IV) in the ferroxidase reaction of an archaeal ferritin, which is based on trapped intermediates obtained with the freeze-quench technique and combination of spectroscopic characterization. We hypothesize that a Fe(IV) intermediate catalyzes oxidation of excess Fe(II) nearby the ferroxidase center.

Keywords: Fe(IV); ferritin; ferroxidase; peroxodiferric; tyrosine radical.

Figure 1

The di‐iron cofactor site of dioxygen‐activating enzymes is different from the di‐iron substrate site of ferritin. (A) The coordinating residues of the di‐iron cofactor site of MMO (PDB 1FYZ) are compared with (B) those of the di‐iron substrate site (the ferroxidase center) of PfFtn (PDB 2JD7). Three main differences are observed (see text). (C) In the di‐iron cofactor site of MMO the peroxodiferric intermediate (two possible molecular structures are shown) decays to intermediate Q, an antiferromagnetically coupled di‐iron Fe(IV) species with a bis‐μ‐oxo diamond core. (D) In ferritin, however, it is believed that the peroxodiferric intermediate directly decays to Fe(III) products.

Figure 2

UV‐visible stopped‐flow spectroscopy suggests the presence of a new intermediate. (A) Two possible pathways proposed for the oxidation of Fe(II) in the ferroxidase center. Pathway 1 occurs in subunits whose sites A and B only are occupied with Fe(II) (A^IIB^IIC⁰ subunits) and pathway 2 occurs in subunits whose sites A, B, and C are occupied with Fe(II) (A^IIB^IIC^II subunits). In the second pathway the highly conserved tyrosine in the ferroxidase center is proposed to provide the fourth electron for a complete reduction of molecular oxygen to water. (B) UV‐visible absorbance spectra of different intermediates recorded for 5 s after the addition of circa 96 Fe(II) per ferritin 24‐mer to PfFtn. Measurements were performed at 47 °C. Concentration of PfFtn was 4.5 μm, which is 10 times less than the concentration of protein used for freeze‐quench experiments. The absorbance at 410 nm disappears after 5 s while the broad absorbance between 500 and 750 increases continuously. (C–F) The UV‐visible absorbance spectra of the intermediates during catalysis of Fe(II) oxidation by PfFtn were obtained using SVD analysis. (C–D) The absorbance spectrum of the intermediates obtained for the addition of 48 Fe(II) per ferritin 24‐mer (4.4 μm), or (E–F) for the addition of 96 Fe(II) per ferritin 24‐mer (15 μm).

Figure 3

Mössbauer spectroscopy provides evidence for the presence of an [Fe(III)‐Fe(IV)] species during catalysis of Fe(II) oxidation in subunits whose sites A, B, and C are occupied with Fe(II). Mössbauer spectrum was recorded for a sample quenched circa 1.0 s after aerobic addition of 48 ⁵⁷Fe(II) per ferritin 24‐mer (45 μm) to apo‐PfFtn. The black solid line (Sum) is the superposition of the simulated spectra. Measurements were performed at 80 K.

Figure 4

The proposed model for the role of the high‐valent Fe(IV) intermediate in the catalysis of Fe(II) oxidation. In pathway 1, when there is no extra Fe(II) nearby the ferroxidase center (site C), the two Fe(II) ions in the ferroxidase center are oxidized via a peroxodiferric intermediate to form the product. The bonding mode of dioxygen in peroxodiferric intermediate is under debate. Recent data based on X‐ray crystallography and Mössbauer spectroscopy suggest a μ‐η1‐η2 bonding mode. In pathway two, when extra Fe(II) is present in the vicinity of the ferroxidase center, first the two Fe(II) ions in the ferroxidase center form the peroxodiferric species. A conformational change occurs due to the presence of Fe(II) at site C. Consequently, the peroxodiferric species rapidly decays as the highly conserved tyrosine provides an electron and a proton. As a result a water molecule and an Fe(IV) intermediate species is formed. The exact binding mode of oxygen in Fe(IV) species is not known (?) and in the picture a terminal oxo group is shown for simplicity. The high‐valent Fe(IV) species then rapidly oxidizes the extra Fe(II) nearby to form a second water molecule and the Fe(III) products. Under reducing conditions the tyrosine radical is possibly reduced by an electron from a yet to be identified redox partner followed by the addition of a proton.