EPR Study of the Mixed-Valent Diiron Sites in Mouse and Herpes Simplex Virus Ribonucleotide Reductases. Effect of the Tyrosyl Radical on Structure and Reactivity of the Diferric Center

Abstract

Reduction of ribonucleotide reductase (EC 1.17.4.1) R2 proteins in a frozen glycerol-buffer solution at 77 K by mobile electrons generated by gamma-irradiation produces EPR-detectable iron sites in mixed-valent Fe(II)/Fe(III) states. The primary EPR signals give information about the ligand arrangement of the diferric form of the iron site, whereas secondary signals observed after annealing of the sample show the effects of structural relaxation. In recombinant metR2 proteins (without free radical) from mouse and herpes virus type 1, the mixed-valent sites trapped at 77 K give rise to axial S = 1/2 EPR spectra with g values in the range 1.79-1.94, observable at temperatures up to 110 K. The spectra are assigned to mu-oxo-bridged dinuclear iron sites. In mouse metR2, the primary EPR spectrum is a mixture of two components. Annealing the R2 samples to 160-170 K transforms the primary EPR signals into rhombic spectra, characterized by gav < 1.8, and observable only below 25 K. These spectra are assigned to partially relaxed forms with a mu-hydroxo bridge, formed by protonation of the oxo bridge. Further annealing at 220 K produces new rhombic EPR spectra, which are closely similar with those observed and found to be stable after chemical reduction at room temperature. The EPR signal of the primary mixed-valent iron site in active mouse R2 protein with a tyrosyl radical also has two components. Both are different from those observed in metR2. In herpes simplex virus type 1 protein R2, one primary mixed-valent component was observed for the met protein. The dose-yield curve for the mixed-valent state in active mouse R2 is sigmoidal in shape, indicating that the tyrosyl radical is reduced by mobile electrons before the iron site. Kinetic experiments on the reduction by dithionite on mouse R2 without and with radical show a significantly enhanced rate for reduction of the iron site in the protein without radical. The results suggest that in active mouse R2 only complete diferric sites with neighboring radicals give rise to the mixed-valent spectra, and that these sites may exist in two structurally distinct forms. The results on the mouse R2 proteins confirm and extend previous results obtained on the Escherichia coli protein R2 showing that the presence of the tyrosyl radical significantly affects not only the structure but also the reactivity of the iron site.

EPR Studies of the Mitochondrial Alternative Oxidase

“…It is typically observed only at very low temperatures due to rapid relaxation. The temperature dependence of the microwave saturation parameter P1 ⁄2 of the signal showed a linear dependence on 1/T as predicted by theory (32,34). (Fig.…”

Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

“…It has been suggested that the hydrogen bond to the μ -oxo bridge could have an influence on the formation of a stable mixed-valence (Fe III Fe II ) cluster. The mixed valence form has been observed by EPR for mouse and herpes simplex virus R2 but not in E. coli R2 or B. cereus R2F [40], [73], [74]. Presently, we were unable to obtain resolved rRaman spectra of the R2F-Mn III 2 -Tyr • form, due to the low radical concentration, stability that the reconstituted protein sample exhibits and presence of the flavin in NrdI with very strong background.…”

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

“…His62, in particular, seems important for proper orientation of the substrate, analogous to Lys127 in MIOX, which is essential for activity (6). With the exception of MIOX, all dinuclear nonheme-iron oxygenases and oxidases studied to date use the fully reduced forms of their cofactors to activate O 2 (32)(33)(34)(35)(36)(37)(38), and the mixedvalent Fe II /Fe III forms are usually only marginally stable in these enzymes (28,39,40). By contrast, MIOX employs the mixedvalent form of its diiron cluster to promote substrate and O 2 activation and accordingly stabilizes this state, allowing its accumulation in 60-70% yield (17 (Fig.…”