Mediation by Indole Analogues of Electron Transfer during Oxygen Activation in Variants of Escherichia coli Ribonucleotide Reductase R2 Lacking the Electron-Shuttling Tryptophan 48
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- 作者：Lana Saleh ; Brian A. Kelch ; Betsy A. Pathickal ; Jeffrey Baldwin ; Brenda A. Ley ; and J. Martin Bollinger ; Jr.
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：May 25, 2004
- 年：2004
- 卷：43
- 期：20
- 页码：5943 - 5952
- 全文大小：129K
- 年卷期：v.43,no.20(May 25, 2004)
- ISSN：1520-4995
文摘
Activation of dioxygen by the carboxylate-bridged diiron(II) cluster in the R2 subunit of classI ribonucleotide reductase from Escherichia coli results in the one-electron oxidation of tyrosine 122(Y122) to a stable radical (Y122
tities/bull.gif">). A key step in this reaction is the rapid transfer of a single electronfrom a near-surface residue, tryptophan 48 (W48), to an adduct between O2 and diiron(II) cluster to generatea readily reducible cation radical (W48+tities/bull.gif">) and the formally Fe(IV)Fe(III) intermediate known as clusterX. Previous work showed that this electron injection step is blocked in the R2 variant with W48 replacedby phenylalanine [Krebs, C., Chen, S., Baldwin, J., Ley, B. A., Patel, U., Edmondson, D. E., Huynh, B.H., and Bollinger, J. M., Jr. (2000) J. Am. Chem. Soc. 122, 12207-12219]. In this study, we show thatsubstitution of W48 with alanine similarly disables the electron transfer (ET) but also permits its chemicalmediation by indole compounds. In the presence of an indole mediator, O2 activation in the R2-W48Avariant produces approximately 1 equiv of stable Y122tities/bull.gif"> and more than 1 equiv of the normal (tities/mgr.gif">-oxo)diiron(III) product. In the absence of a mediator, the variant protein generates primarily altered Fe(III)products and only one-fourth as much stable Y122tities/bull.gif"> because, as previously reported for R2-W48F, mostof the Y122tities/bull.gif"> that is produced decays as a consequence of the inability of the protein to mediate reductivequenching of one of the two oxidizing equivalents of the initial diiron(II)-O2 complex. Mediation of ETis effective in W48A variants containing additional substitutions that also impact the reaction mechanismor outcome. In the reaction of R2-W48A/F208Y, the presence of mediator suppresses formation of theY208-derived diiron(III)-catecholate product (which is predominant in R2-F208Y in the absence ofreductants) in favor of Y122tities/bull.gif">. In the reaction of R2-W48A/D84E, the presence of mediator affects theoutcome of decay of the peroxodiiron(III) intermediate known to accumulate in D84E variants, increasingthe yield of Y122tities/bull.gif"> by as much as 2.2-fold to a final value of 0.75 equiv and suppressing formation of a490 nm absorbing product that results from decay of the two-electron oxidized intermediate in the absenceof a functional ET apparatus.
tities/bull.gif">). A key step in this reaction is the rapid transfer of a single electronfrom a near-surface residue, tryptophan 48 (W48), to an adduct between O2 and diiron(II) cluster to generatea readily reducible cation radical (W48+tities/bull.gif">) and the formally Fe(IV)Fe(III) intermediate known as clusterX. Previous work showed that this electron injection step is blocked in the R2 variant with W48 replacedby phenylalanine [Krebs, C., Chen, S., Baldwin, J., Ley, B. A., Patel, U., Edmondson, D. E., Huynh, B.H., and Bollinger, J. M., Jr. (2000) J. Am. Chem. Soc. 122, 12207-12219]. In this study, we show thatsubstitution of W48 with alanine similarly disables the electron transfer (ET) but also permits its chemicalmediation by indole compounds. In the presence of an indole mediator, O2 activation in the R2-W48Avariant produces approximately 1 equiv of stable Y122tities/bull.gif"> and more than 1 equiv of the normal (tities/mgr.gif">-oxo)diiron(III) product. In the absence of a mediator, the variant protein generates primarily altered Fe(III)products and only one-fourth as much stable Y122tities/bull.gif"> because, as previously reported for R2-W48F, mostof the Y122tities/bull.gif"> that is produced decays as a consequence of the inability of the protein to mediate reductivequenching of one of the two oxidizing equivalents of the initial diiron(II)-O2 complex. Mediation of ETis effective in W48A variants containing additional substitutions that also impact the reaction mechanismor outcome. In the reaction of R2-W48A/F208Y, the presence of mediator suppresses formation of theY208-derived diiron(III)-catecholate product (which is predominant in R2-F208Y in the absence ofreductants) in favor of Y122tities/bull.gif">. In the reaction of R2-W48A/D84E, the presence of mediator affects theoutcome of decay of the peroxodiiron(III) intermediate known to accumulate in D84E variants, increasingthe yield of Y122tities/bull.gif"> by as much as 2.2-fold to a final value of 0.75 equiv and suppressing formation of a490 nm absorbing product that results from decay of the two-electron oxidized intermediate in the absenceof a functional ET apparatus.