Redox-dependent structural coupling between the α2 and β2 subunits in E. coli ribonucleotide reductase

Ribonucleotide reductase (RNR) is essential for DNA synthesis in all organisms. In E. coli, the class Ia RNR forms a transient α2β2 complex during catalysis. The β2 subunit contains a tyrosyl radical cofactor that initiates the reduction of ribonucleotides in α2 through a proton-coupled electron transfer mechanism. Prior research has shown that this process is regulated by allosteric effectors like ATP. However, the structural coupling between α2 and β2 subunits during inhibition or substrate binding remains poorly understood. No prior work had resolved the exact conformational changes in the α2β2 complex under turnover or inhibitory conditions. This gap motivated the use of reaction-induced FT-IR spectroscopy to explore subunit-specific structural responses. The study aimed to clarify how dATP and hydroxyurea affect the structural dynamics of the α2β2 complex. Understanding these interactions could provide insights into the enzyme’s regulation and inhibition mechanisms.

The purpose of this study was to investigate the structural coupling between α2 and β2 subunits in E. coli ribonucleotide reductase during catalysis and inhibition. The researchers sought to determine how substrate binding and allosteric effectors influence the conformational dynamics of the α2β2 complex. They used isotopic labeling and reaction-induced FT-IR spectroscopy to track subunit-specific structural changes. The study aimed to identify the role of tyrosyl radical and diferric cofactor in mediating structural communication between subunits. By analyzing the effects of dATP and hydroxyurea, the researchers intended to clarify how these inhibitors modulate the enzyme’s function. The work also aimed to detect conformational changes in β2 tyrosines during complex formation. The goal was to demonstrate the utility of FT-IR spectroscopy in studying enzyme dynamics.

The researchers used reaction-induced FT-IR spectroscopy to monitor structural changes in the α2β2 complex. Isotopic chimeras, (13)Cα2β2 and α2(13)Cβ2, were prepared to identify subunit-specific contributions. Spectra were collected after mixing α2 and β2 with CDP and ATP. The study included the use of dATP and hydroxyurea as inhibitors to observe their effects on the complex. Amide I and II bands were analyzed to detect structural rearrangements in α2 and β2. (2)H4 labeling of β2 tyrosines was used to track the tyrosyl radical and its reduction by hydroxyurea. HU editing was applied to distinguish Y122O• and Y122OH states in the complex. The spectral data were interpreted to identify conformational changes in β2 tyrosines during complex formation.

The study revealed that dATP binding reduced the contribution of amide I bands from β strands and disordered structures in β2. Hydroxyurea (HU) caused structural changes in both α2 and β2 subunits, particularly in tyrosine residues. The tyrosyl radical (Y122O•) and its hydroxyl form (Y122OH) were detected in the α2β2 complex using (2)H4 labeling. D84, a ligand to the diferric cluster, was also observed in the complex. A conformational rearrangement was identified at an additional β2 tyrosine(s), Yx, in the α2β2/CDP/ATP complex. The amide I and II bands indicated structural coupling between α2 and β2 during turnover. The results showed that HU-mediated reduction of Y122O• is associated with α2 structural changes. The study confirmed the utility of FT-IR spectroscopy in tracking enzyme dynamics.

The findings suggest that structural coupling between α2 and β2 subunits is crucial for the function of E. coli ribonucleotide reductase. The study demonstrates that dATP and hydroxyurea modulate the structural dynamics of the α2β2 complex. The tyrosyl radical and diferric cofactor in β2 play a key role in mediating these changes. The results support the hypothesis that conformational rearrangements in β2 tyrosines are linked to enzyme inhibition. The use of isotopic labeling and FT-IR spectroscopy proved effective in identifying subunit-specific structural changes. The study provides evidence for a redox-dependent mechanism in the α2β2 complex. The findings align with the authors’ claim that structural communication between subunits is essential for catalytic function. The work highlights the importance of spectroscopic methods in studying enzyme regulation.