title: 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.
authors.affiliation: School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.
authors.forename: Adam R
authors.initials: AR
authors.lastname: Offenbacher
authors.forename: R Atlee
authors.initials: RA
authors.lastname: Watson
authors.forename: Cynthia V
authors.initials: CV
authors.lastname: Pagba
authors.forename: Bridgette A
authors.initials: BA
authors.lastname: Barry
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D012264:Ribonucleotide Reductases