Radical-Translocation Intermediates and Hurdling of Pathway Defects in 鈥淪uper-oxidized鈥?(MnIV/FeIV) Chlamydia trachomatis Ribonucleotide Reductase
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- 作者：Laura M. K. Dassama ; Wei Jiang ; Paul T. Varano ; Maria-Eirini Pandelia ; Denise A. Conner ; Jiajia Xie ; J. Martin Bollinger ; Jr. ; Carsten Krebs
- 刊名：The Journal of the American Chemical Society
- 出版年：2012
- 出版时间：December 19, 2012
- 年：2012
- 卷：134
- 期：50
- 页码：20498-20506
- 全文大小：433K
- 年卷期：v.134,no.50(December 19, 2012)
- ISSN：1520-5126
文摘
A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y鈥?/sup>) or a MnIV/FeIII cluster in its 尾 subunit to oxidize a cysteine residue 35 脜 away in its 伪 subunit, generating a thiyl radical that abstracts hydrogen (H鈥?/sup>) from the substrate. With either oxidant, the inter-subunit 鈥渉ole-transfer鈥?or 鈥渞adical-translocation鈥?(RT) process is thought to occur by a 鈥渉opping鈥?mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The MnIV/FeIII cofactor of Chlamydia trachomatis RNR assembles via a MnIV/FeIV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when 伪 is present, accumulating radicals with EPR spectra characteristic of Y鈥?/sup>鈥檚. The dependence of Y鈥?/sup> accumulation on the presence of substrate suggests that RT within this 鈥渟uper-oxidized鈥?enzyme form is gated by the protein, and the failure of a 尾 variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y鈥?/sup>(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant 尾 proteins having pathway substitutions rendering them inactive in their MnIV/FeIII states can generate the pathway Y鈥?/sup>鈥檚 in their MnIV/FeIV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the 鈥渉urdling鈥?of engineered defects in the RT pathway.