Hydrogen Bond Network between Amino Acid Radical Intermediates on the Proton-Coupled Electron Transfer Pathway ofE. coliα2 Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-β, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. 2H electron–nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed.

Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm

“…Our original multi-frequency EPR analysis of NH 2 Y 730 •-RNR 25 was interpreted to favor a pyramidal (RNH 2 Y•) structure, whereas later quantum chemical calculations on a geometry optimized structure of NH 2 Y 730 •-α2 and NH 2 Y 731 •-α2 predicted an almost planar conformation of the trapped NH 2 Y•s in the protein environment of α2. 26, 27 To obtain a direct measurement of the amino protons hyperfine coupling (hfc) and their orientations, we synthesized D 6 -NH 2 Y 731 -α2 and generated the D 6 -NH 2 Y 731 •-α2 in the presence of β2, substrate, and effector. This strategy permits a distinction between the resonances of the amino protons and all resonances of the internal 1 Hs in NH 2 Y•.…”

Strong cooperative effects between π-hole and dihydrogen bonds interactions: a computational study

“…The biological activities of most macromolecules like proteins and polypeptides in living cells are largely controlled by the NCIs. Hence, it is not surprising that extensive research interest has focused on proper understanding of the importance of these interactions in last decades [4][5][6][7]. Of the various NCIs, the hydrogen bond (HB) is undoubtedly the most widely studied and best understood case.…”

Extension of the fragment molecular orbital method to treat large open-shell systems in solution

“…As an application, the ribonucleotide reductase (PDB: 2X0X) [37,38] was studied. The PDB structure was protonated in AMBER [46], optimized, and an NPT molecular dynamics was performed in explicit water for 1 ns using AMBER99 force field [47] as implemented in AMBER 12 [46].…”

“…FMO is applied to study the spin density and radical stabilization factors in the doublet spin state of the ribonucleotide reductase (PDB: 2X0X) [37,38].…”