The Core Structure of X Generated in the Assembly of the Diiron Cluster of Ribonucleotide Reductase: 17O2 and H217O ENDOR

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• 作者：Doug Burdi ; Jean-Paul Willems ; Pam Riggs-Gelasco ; William E. Antholine ; JoAnne Stubbe ; and Brian M. Hoffman
• 刊名：Journal of the American Chemical Society
• 出版年：1998
• 出版时间：December 16, 1998
• 年：1998
• 卷：120
• 期：49
• 页码：12910 - 12919
• 全文大小：159K
• 年卷期：v.120,no.49(December 16, 1998)
• ISSN：1520-5126

文摘

The intermediate, designated X, formed during the self-assembly reaction of the tyrosyl radical/-oxo-bridged diferric cofactor in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) is directlyinvolved in the oxidation of Y122 to the catalytically essential ·Y122. Earlier rapid freeze-quench (RFQ)Q-band ENDOR studies led to the formulation of X as a spin-coupled Fe^III/Fe^IV center, with an S = ¹/₂ groundstate, and showed that X contains a single terminal aqua ligand (water molecule or 2-fold disordered hydroxyl)bound to Fe^III but does not contain an hydroxyl bridge. That ENDOR data, coupled with RFQ-EXAFS data,plus the strong spin coupling between the iron ions constrain the structure of X to a di- or tribridged specieswhose inorganic core (defined as iron and exogenous ligands) contains the [(H_xO)Fe^IIIOFe^IV] fragment. Todetermine whether the core contains a second oxo bridge and to establish the fate of the atoms derived fromO₂, we have now performed CW and pulsed Q-band ¹⁷O ENDOR experiments on samples of X prepared inboth H₂¹⁷O and ¹⁷O₂, using a uniformly ¹⁵N-labeled protein, [U-¹⁵N]-R2. These measurements, along withkinetic studies on the formation of X in both wild-type and Y122F R2, as monitored by both ENDOR andS-band EPR spectroscopies, reveal that X contains two oxygen atoms. Both are initially derived from O₂, withone present as a -oxo bridge and one as the terminal aqua ligand; with time the latter of these atoms exchangeswith solvent. These and our previous studies indicate that X does not contain a di--oxo- or -oxo,hydroxo-bridged core structure. A structure for X is proposed that contains a single oxo bridge, one terminal aqualigand bound to the Fe^III, and one or two additional mono-oxo bridges provided by the carboxylate oxygensof E115 and/or E238. In addition, the time course of the formation of X in the presence of ¹⁷O₂ providesimportant insights into the dynamics of cluster assembly.