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.

Hydroxyurea—The Good, the Bad and the Ugly

“…DNA synthesis and repair depend heavily on the activity of ribonucleotide reductases (RNRs)—enzymes that utilize proton-coupled electron transfer (PCET) reactions to catalyze the reduction of ribonucleotides to deoxyribonucleotides [ 34 , 35 ]. The reaction catalyzed by RNR is an irreversible and crucial step in the process of building the free nucleotide pool—the substrates needed for DNA replication as well as repair.…”

“…It has been proposed that the reaction involving proton transfer is facilitated by an extensive proton wire composed of a hydrogen-bonded network that includes aspartate and glutamates. The reaction in which a proton and electron are donated to a radical site in the RNR subunit β2 seems to be more direct and independent of the proton wire [ 35 ].…”

“…This binding allows for substrate–effector interaction. ( F ) Hydroxyurea inhibits PCET within RNR by proton-coupled electron transfer, most probably mediated by a hydrogen-bonded proton wire [ 35 ]. The images in ( A , B ) were generated using Mol*Viewer [ 46 ].…”

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

“…Model for the inhibition reaction of the E. coli β 2 Y122· by the clinical drug HU via PCET. Reduction of Y122· by HU is facilitated by a long-range, carboxylate containing proton wire (orange arrow) . During reduction, the tyrosyl radical changes conformation so that it forms a hydrogen bonding interaction with D84, a ligand to the β 2 diferric cluster.…”

“…During reduction, the tyrosyl radical changes conformation so that it forms a hydrogen bonding interaction with D84, a ligand to the β 2 diferric cluster. Adapted in part with permission from ref . Copyright 2020 American Chemical Society.…”