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Mechanistic Implications for the Formation of the Diiron Cluster in Ribonucleotide Reductase Provided by Quantitative EPR Spectroscopy

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posted on 2003-06-28, 00:00 authored by Brad S. Pierce, Timothy E. Elgren, Michael P. Hendrich
The small subunit of Escherichia coli ribonucleotide reductase (R2) is a homodimeric (ββ) protein, in which each β-peptide contains a diiron cluster composed of two inequivalent iron sites. R2 is capable of reductively activating O2 to produce a stable tyrosine radical (Y122•), which is essential for production of deoxyribonucleotides on the larger R1 subunit. In this work, the paramagnetic MnII ion is used as a spectroscopic probe to characterize the assembly of the R2 site with EPR spectroscopy. Upon titration of MnII into samples of apoR2, we have been able to quantitatively follow three species (aquaMnII, mononuclear MnIIR2, and dinuclear Mn2IIR2) and fit each to a sequential two binding site model. As previously observed for FeII binding within apoR2, one of the sites has a greater binding affinity relative to the other, K1 = (5.5 ± 1.1) × 105 M-1 and K2 = (3.9 ± 0.6) × 104 M-1, which are assigned to the B and A sites, respectively. In multiple titrations, only one dinuclear Mn2IIR2 site was created per homodimer of R2, indicating that only one of the two β-peptides of R2 is capable of binding MnII following addition of MnII to apoR2. Under anaerobic conditions, addition of only 2 equiv of FeII to R2 (Fe2IIR2) completely prevented the formation of any bound MnR2 species. Upon reaction of this sample with O2 in the presence of MnII, both Y122• and Mn2IIR2 were produced in equal amounts. Previous stopped-flow absorption spectroscopy studies have indicated that apoR2 undergoes a protein conformational change upon binding of metal (Tong et al. J. Am. Chem. Soc. 1996, 118, 2107−2108). On the basis of these observations, we propose a model for R2 metal incorporation that invokes an allosteric interaction between the two β-peptides of R2. Upon binding the first equiv of metal to a β-peptide (βI), the aforementioned protein conformational change prevents metal binding in the adjacent β-peptide (βII) approximately 25 Å away. Furthermore, we show that metal incorporation into βII occurs only during the O2 activation chemistry of the βI-peptide. This is the first direct evidence of an allosteric interaction between the two β-peptides of R2. Furthermore, this model can explain the generally observed low Fe occupancy of R2. We also demonstrate that metal uptake and this newly observed allosteric effect are buffer dependent. Higher levels of glycerol cause loss of the allosteric effect. Reductive cycling of samples in the presence of MnII produced a novel mixed metal FeIIIMnIIIR2 species within the active site of R2. The magnitude of the exchange coupling (J) determined for both the Mn2IIR2 and FeIIIMnIIIR2 species was determined to be −1.8 ± 0.3 and −18 ± 3 cm-1, respectively. Quantitative spectral simulations for the FeIIIMnIIIR2 and mononuclear MnIIR2 species are provided. This work represents the first instance where both X- and Q-band simulations of perpendicular and parallel mode spectra were used to quantitatively predict the concentration of a protein bound mononuclear MnII species.

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