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
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- 作者：Danny Yun ; Carsten Krebs ; Govind P. Gupta ; David F. Iwig ; Boi Hanh Huynh ; and J. Martin Bollinger ; Jr.
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：January 22, 2002
- 年：2002
- 卷：41
- 期：3
- 页码：981 - 990
- 全文大小：127K
- 年卷期：v.41,no.3(January 22, 2002)
- ISSN：1520-4995
文摘
The kinetics and mechanism of formation of the tyrosyl radical and 
-(oxo)diiron(III) clusterin the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorptionand 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 ofY177 by one of these intermediates to form the stable radical, with concomitant or subsequent formationof the adjacent -(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediatespectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction ofthe Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably theY177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalentto the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative toother steps in the reaction, the present data indicate that the transfer of this reducing equivalent is notrate-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 givethe 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 stepoverall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer ofthe reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions aremechanistically similar.
-(oxo)diiron(III) clusterin the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorptionand 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 ofY177 by one of these intermediates to form the stable radical, with concomitant or subsequent formationof the adjacent -(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediatespectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction ofthe Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably theY177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalentto the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative toother steps in the reaction, the present data indicate that the transfer of this reducing equivalent is notrate-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 givethe 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 stepoverall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer ofthe reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions aremechanistically similar.