E. coli Ribonucleotide Reductase ß2 Subunit Inactivation by Triapine Occurs through Binding of a Triapine-Fe(II) Adduct.

Ribonucleotide reductase (RNR), which supplies the building blocks for DNA biosynthesis and its repair, has been linked to human diseases and is emerging as a therapeutic target. Here, we present a mechanistic investigation of triapine (3AP), a clinically relevant small molecule that inhibits the tyrosyl radical within the RNR ß2 subunit. Solvent kinetic isotope effects reveal that proton transfer is not rate-limiting for inhibition of Y122· of E. coli RNR ß2 by the pertinent 3AP-Fe(II) adduct. Vibrational spectroscopy further demonstrates that unlike inhibition of the ß2 tyrosyl radical by hydroxyurea, a carboxylate containing proton wire is not at play. Binding measurements reveal a low nanomolar affinity (Kd ∼ 6 nM) of 3AP-Fe(II) for ß2. Taken together, these data should prompt further development of RNR inactivators based on the triapine scaffold for therapeutic applications.

Redox-dependent structural coupling between the α2 and ß2 subunits in E. coli ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient α2ß2 complex forms when a ribonucleotide substrate, such as CDP, binds to the α2 subunit. A tyrosyl radical (Y122O•)-diferric cofactor in ß2 initiates substrate reduction in α2 via a long-distance, proton-coupled electron transfer (PCET) process. Here, we use reaction-induced FT-IR spectroscopy to describe the α2ß2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing E. coli α2 and ß2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, (13)Cα2ß2 and α2(13)Cß2, were used to define subunit-specific structural changes. Mixing of α2 and ß2 under turnover conditions yielded amide I (C═O) and II (CN/NH) bands, derived from each subunit. The addition of the inhibitor, dATP, resulted in a decreased contribution from amide I bands, attributable to ß strands and disordered structures. Significantly, HU-mediated reduction of Y122O• was associated with structural changes in α2, as well as ß2. To define the spectral contributions of Y122O•/Y122OH in the quaternary complex, (2)H4 labeling of ß2 tyrosines and HU editing were performed. The bands of Y122O•, Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated ß2, were observed in the α2ß2 complex. These spectra also provide evidence for a conformational rearrangement at an additional ß2 tyrosine(s), Yx, in the α2ß2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes.