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¹⁹F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase.

ABSTRACT: Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α₂β₂ enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β₂, nucleotide reduction in α₂. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y₃₅₆(β) and Y₇₃₁(α) across the α/β interface. Using 2,3,5-F₃Y₁₂₂-β₂ with 3,5-F₂Y₇₃₁-α₂, GDP (substrate) and TTP (allosteric effector), a Y₃₅₆^• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz ¹⁹F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y₃₅₆^• and the ¹⁹F nuclei of 3,5-F₂Y₇₃₁ in this RNR mutant. Similar experiments with the double mutant E₅₂Q/F₃Y₁₂₂-β₂ were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F₂Y₇₃₁. Remarkably, one conformation is consistent with 3,5-F₂Y₇₃₁ within the H-bond distance to Y₃₅₆^•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y₃₅₆ and Y₇₃₁. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

SUBMITTER: Meyer A

PROVIDER: S-EPMC9248007 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tyrosyl radicals play essential roles in biological proton-coupled electron transfer (PCET) reactions. Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides and is vital in DNA replication in all organisms. Class Ia RNRs consist of ?2 and ?2 homodimeric subunits. In class Ia RNR, such as the E. coli enzyme, an essential tyrosyl radical (Y122O(•))-diferric cofactor is located in ?2. Although Y122O(•) is extremely stable in free ?2, Y122O(•) is highly reactive in the quaternary substrate-?2?2 complex and serves as a radical initiator in catalytic PCET between ?2 and ?2. In this report, we investigate the structural interactions that control the reactivity of Y122O(•) in a model system, isolated E. coli ?2. Y122O(•) was reduced with hydroxyurea (HU), a radical scavenger that quenches the radical in a clinically relevant reaction. In the difference FT-IR spectrum, associated with this PCET reaction, amide I (CO) and amide II (CN/NH) bands were observed. Specific (13)C-labeling of the tyrosine C1 carbon assigned a component of these bands to the Y122-T123 amide bond. Comparison to density functional calculations on a model dipeptide, tyrosine-threonine, and structural modeling demonstrated that PCET is associated with a Y122 rotation and a 7.2 Å translation of the Y122 phenolic oxygen. To test for the functional consequences of this structural change, a proton inventory defined the origin of the large solvent isotope effect (SIE = 16.7 ± 1.0 at 25 °C) on this reaction. These data suggest that the one-electron, HU-mediated reduction of Y122O(•) is associated with two, rate-limiting (full or partial) proton transfer reactions. One is attributable to HU oxidation (SIE = 11.9, net H atom transfer), and the other is attributable to coupled, hydrogen-bonding changes in the Y122O(•)-diferric cofactor (SIE = 1.4). These results illustrate the importance of redox-linked changes to backbone and ring dihedral angles in high potential PCET and provide evidence for rate-limiting, redox-linked hydrogen-bonding interactions between Y122O(•) and the iron cluster.

Project description:The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein-O2 complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H2O2 and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and β-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and β-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferric intermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferric intermediates decay with measurable rates at temperatures above 170 K with appreciable solvent kinetic isotope effects. The hydroperoxoferric intermediate of β-chains converts to the S = 3/2 Cpd I, which in turn decays to an electron paramagnetic resonance (EPR)-silent product at temperature above 220 K. For all the other hemoproteins studied, cryoannealing of the hydroperoxo intermediate directly yields an EPR-silent majority product. In each case, a second follow-up 77 K γ-irradiation of the annealed samples yields low-spin EPR signals characteristic of cryoreduced ferrylheme (compound II, Cpd II). This indicates that in general the hydroperoxoferric intermediates relax to Cpd I during cryoanealing at low temperatures, but when this state is not captured by reaction with a bound substrate, it is reduced to Cpd II by redox-active products of radiolysis.

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¹⁹F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase.

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19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase.

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¹⁹F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase.