A Proton Wire Mediates Proton Coupled Electron Transfer from Hydroxyurea and Other Hydroxamic Acids to Tyrosyl Radical in Class Ia Ribonucleotide Reductase

Abstract

Proton-coupled electron transfer (PCET) is fundamental to many important biological reactions, including solar energy conversion and DNA synthesis. For example, class Ia ribonucleotide reductases (RNRs) contain a tyrosyl radical-diiron cofactor with one aspartate ligand, D84. The tyrosyl radical, Y122^•, in the β2 subunit acts as a radical initiator and oxidizes an active site cysteine in the α2 subunit. A transient quaternary α2/β2 complex is induced by substrate and effector binding. The hydroxamic acid, hydroxyurea (HU), reduces Y122^• in a PCET reaction involving an electron and proton. This reaction is associated with the loss of activity, a conformational change at Y122, and a change in hydrogen bonding to the Fe1 ligand, D84. Here, we use isotopic labeling, solvent isotope exchange, proton inventories, and reaction-induced Fourier transform infrared (RIFT-IR) spectroscopy to show that the PCET reactions of hydroxamic acids are associated with a characteristic spectrum, which is assignable to electrostatic changes at nonligating aspartate residues. Notably, RIFT-IR spectroscopy reveals this characteristic spectrum when the effects of HU, hydroxylamine, and N-methylhydroxylamine are compared. A large solvent isotope effect is observed for each of the hydroxamic acid reactions, and proton inventories predict that the reactions are associated with the transfer of multiple protons in the transition state. The reduction of Y122^• with 4-methoxyphenol does not lead to these characteristic carboxylate shifts and is associated with only a small solvent isotope effect. In addition to studies of the effects of hydroxamic acids on β2 alone, the reactions involving the quaternary α2β2 complex were also investigated. HU treatment of the quaternary complex, α2/β2/ATP/CDP, leads to a similar carboxylate shift spectrum, as observed with β2 alone. The use of globally labeled ¹³C chimeras (¹³C α2, ¹³C β2) confirms the assignment. Because the spectrum is sensitive to ¹³C β2 labeling, but not ¹³C α2 labeling, the quaternary complex spectrum is assigned to electrostatic changes in β2 carboxylate groups. Examination of the β2 X-ray structure reveals a hydrogen-bonded network leading from the protein surface to Y122. This predicted network includes nonligating aspartates, glutamate ligands to the iron cluster, and predicted crystallographically resolved water molecules. The network is similar when class Ia RNR structures from Escherichia coli, human, and mouse are compared. We propose that the PCET reactions of hydroxamic acids are mediated by a hydrogen-bonded proton wire in the β2 subunit.