Redox-Dependent Structural Coupling between the α2 and β2 Subunits in E. coli Ribonucleotide Reductase

Abstract

Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient α2β2 complex forms when a ribonucleotide substrate, such as CDP, binds to the α2 subunit. A tyrosyl radical (Y122O•)-diferric cofactor in β2 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 α2β2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing E. coli α2 and β2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, (13)Cα2β2 and α2(13)Cβ2, were used to define subunit-specific structural changes. Mixing of α2 and β2 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 β strands and disordered structures. Significantly, HU-mediated reduction of Y122O• was associated with structural changes in α2, as well as β2. To define the spectral contributions of Y122O•/Y122OH in the quaternary complex, (2)H4 labeling of β2 tyrosines and HU editing were performed. The bands of Y122O•, Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated β2, were observed in the α2β2 complex. These spectra also provide evidence for a conformational rearrangement at an additional β2 tyrosine(s), Yx, in the α2β2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes.

3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound

“…These structural results also offer insight into inhibition of RNR by the diphosphate and triphosphate forms of the therapeutics clofarabine (ClFDP and ClFTP), cladribine (ClADP and ClATP), and fludarabine (FlUTP) that are known to impart α hexamers with enhanced stability ( Aye and Stubbe, 2011 ; Aye et al, 2012 ; Wisitpitthaya et al, 2016 ). Given that the well-studied RNR inhibitors gemcitabine-diphosphate and hydroxyurea are known to target the active site on α and the tyrosyl radical on β 2 , respectively ( van der Donk et al, 1998 ; Offenbacher et al, 2014 ), the possibility that some RNR inhibitors might act by targeting the allosteric activity site on α was intriguing to investigate. ClF and ClA are cytotoxic in a cell line expressing wild-type α, whereas a cell line expressing an α mutation in its allosteric activity site (Asp57Asn) is resistant to the drugs ( Wisitpitthaya et al, 2016 ), consistent with the allosteric activity site being targeted by these molecules.…”

E. coli Ribonucleotide Reductase β2 Subunit Inactivation by Triapine Occurs through Binding of a Triapine–Fe(II) Adduct

“…A mechanistic understanding of 3AP inhibition at the molecular level and its binding interaction with β 2 is still largely lacking, however, with support for mechanisms involving the production of radical oxygen species (ROS) , and by direct inhibition of the tyrosyl radical in human β 2 . Here, we study inhibition of E. coli β 2 by 3AP using in situ Fourier transform infrared (FTIR) difference spectroscopy, − solvent kinetic isotope effects (SIE) as employed previously for mechanistic investigations of HU, and binding studies to bolster the hypothesis that RNR inhibition by triapine occurs through a tight binding of Fe­(II)-loaded 3AP to β 2 .…”

Hydrogen Bonds and Electrostatic Environment of Radical Intermediates in Ribonucleotide Reductase Ia

“…127 However, a conformeric change at a Y has been reported by infrared (IR) spectroscopy in β. 128 They compared the non-oxidized to the oxidized state within minutes reaction time. 128 IR spectroscopy normally need well characterized ring dihedrals and backbone dihedrals in order to assign IR shifts precisely (cf.…”

“…128 They compared the non-oxidized to the oxidized state within minutes reaction time. 128 IR spectroscopy normally need well characterized ring dihedrals and backbone dihedrals in order to assign IR shifts precisely (cf. Figure 1-12B).…”

“…Figure 1-12B). [129][130][131] Barry et al propose based on their Y,T di-peptide model [129][130][131] a conformeric change of Y122•; 128 however, the apparent contradiction between the ring dihedral from EPR spectral simulation (43°) 132 and their assigned ring dihedral (80°) is currently not resolved. 133 Generally, information on ring dihedrals within the PCET of RNR is reported only at one additional position.…”