TY - JOUR
T1 - Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse
AU - Yun, Danny
AU - Krebs, Carsten
AU - Gupta, Govind P.
AU - Iwig, David F.
AU - Huynh, Boi Hanh
AU - Bollinger, J. Martin
PY - 2002/1/22
Y1 - 2002/1/22
N2 - The kinetics and mechanism of formation of the tyrosyl radical and μ-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent μ-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (kobs = 13 ± 3 s-1 at 5 °C and 0.95 mM O2) and the decay of the intermediate to give the Y177 radical (kobs = 5 ± 2 s-1) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)aq (kobs = 0.29 ± 0.03 s-1), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.
AB - The kinetics and mechanism of formation of the tyrosyl radical and μ-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent μ-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (kobs = 13 ± 3 s-1 at 5 °C and 0.95 mM O2) and the decay of the intermediate to give the Y177 radical (kobs = 5 ± 2 s-1) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)aq (kobs = 0.29 ± 0.03 s-1), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.
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U2 - 10.1021/bi011797p
DO - 10.1021/bi011797p
M3 - Article
C2 - 11790122
AN - SCOPUS:0037154128
SN - 0006-2960
VL - 41
SP - 981
EP - 990
JO - Biochemistry
JF - Biochemistry
IS - 3
ER -