Kinetic model for reversible radical transfer in ribonucleotide reductase

Abstract

The enzyme ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleotides to deoxynucleotides, is vital for DNA synthesis, replication, and repair in all living organisms. Its mechanism requires long-range radical translocation over ∼32 Å through two protein subunits and the intervening aqueous interface. Herein, a kinetic model is designed to describe reversible radical transfer in Escherichia coli RNR. This model is based on experimentally studied photoRNR systems that allow the photochemical injection of a radical at a specific tyrosine residue, Y356, using a photosensitizer. The radical then transfers across the interface to another tyrosine residue, Y731, and continues until it reaches a cysteine residue, C439, which is primed for catalysis. This kinetic model includes radical injection, an off-pathway sink, radical transfer between pairs of residues along the pathway, and the conformational flipping motion of Y731 at the interface. Most of the input rate constants for this kinetic model are obtained from previous experimental measurements and quantum mechanical/molecular mechanical free-energy simulations. Ranges for the rate constants corresponding to radical transfer across the interface are determined by fitting to the experimentally measured Y356 radical decay times in photoRNR systems. This kinetic model illuminates the time evolution of radical transport along the tyrosine and cysteine residues following radical injection. Further analysis identifies the individual rate constants that may be tuned to alter the timescale and probability of the injected radical reaching C439. The insights gained from this kinetic model are relevant to biochemical understanding and protein-engineering efforts with potential pharmacological implications.

Kinetic Diagram Analysis: A Python Library for Calculating Steady-State Observables of Biochemical Systems Analytically

“…Proton leakage cycle dominance demonstrates the competition between symport and uniport cycles, where symport cycles have to compete with fast alternating access allowing for inefficient transport as suggested by the original study. 32 This demonstrates the sensitivity of symport pathways to leakage when the alternating access rates are a similar order of magnitude as the substrate off-rates (for R off = 100 the drug off-rates are 10 1 − 10 3 s −1 ), resulting in less efficient cotransport and smaller drug fluxes.…”

“…To demonstrate the use of KDA we are fo- cusing on the biophysics of secondary active transporters, [27][28][29] an area covered originally in Hill's work 11,12 with many recent applications of kinetic models. [1][2][3][4][5][6][30][31][32][33] These membrane proteins use the free energy stored in a transmembrane ionic gradient to drive energetically uphill transport of a substrate (another ion or small molecule) by alternating between multiple protein conformations in a manner that is coupled to ion and substrate binding. Secondary active transporters are prime examples of free energy transduction in biology 12 and serve as systems to study non-equilibrium behavior at the molecular scale.…”

Direct Proton-Coupled Electron Transfer between Interfacial Tyrosines in Ribonucleotide Reductase

“…Such insights will most likely rely on kinetic modeling, as performed in our previous work for photosensitized RNRs within the α subunit using a combination of computational and experimental inputs. 23…”

“…Furthermore, these simulations do not provide information about the other PCET reactions along the pathway or insights into the global thermodynamics of radical propagation in RNR. Such insights will most likely rely on kinetic modeling, as performed in our previous work for photosensitized RNRs within the α subunit using a combination of computational and experimental inputs …”

“…We conducted QM/MM finite temperature string simulations with umbrella sampling , to calculate the free energy surfaces for both water-mediated and direct PCET between Y356 and Y731. The initial conformations with Y731 in the flipped conformation were obtained from the classical MD simulations described above and in the SI, and the initial conformations with Y731 in the stacked conformation were obtained from our previous MD simulations with a restraint on the Y731 • sidechain . The QM region was described with the ωB97X-D functional and the 6-31+G** basis set, − and the MM region was described with the AMBER ff14SB force field. − This level of theory for the QM region was shown to be consistent with multireference complete active space self-consistent field calculations with second-order perturbative corrections (CASSCF+NEVPT2) for PCET between two tyrosine molecules in our previous work .…”