Understanding the Electon Paramagnetic Resonance Parameters of Protein-Bound Glycyl Radicals

Abstract

The number of enzymes that require a glycyl-based radical for their function is growing. Here, we provide systematic quantum-chemical studies of spin-density distributions, electronic g-tensors, and hyperfine couplings of various models of protein-bound glycyl radicals. Similarly to what is found in a companion paper on N-acetylglycyl, the small g-anisotropy for this delocalized, unsymmetrical system presents appreciable challenges to state-of-the-art computational methodology. This pertains to the quality of structure optimization, as well as to the choice of the spin-orbit Hamiltonian and the gauge origin of the magnetic vector potential. Environmental effects due to hydrogen bonding are complicated and depend in a subtle fashion on the different intramolecular hydrogen bonding for different conformations of the radical. Indeed, the conformation has the largest overall effect on the computed g-tensors (less so on the hyperfine tensors). This is discussed in the context of different g-tensors obtained by recent high-field electron paramagnetic resonance (EPR) measurements for three different enzymes. On the basis of results of a parallel calibration study for N-acetylglycyl, it is suggested that the glycyl radical observed for E. coli anaerobic RNR may have a fully extended conformation, which differs from those of the corresponding radicals in pyruvate formate-lyase or benzylsuccinate synthase.

Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme

“…The location of peptide binding and the AdoMet C5Ј to G 734 C ␣ distance are consistent with the substratebinding sites observed in the other AdoMet radical enzyme structures (5,8). Additionally, the torsional angles of G 734 as modeled in this structure are similar to both those seen in the PFL structure (18,21) and those predicted for the PFL glycyl radical by quantumchemical studies of the EPR parameters of the PFL radical (36). The pept-AE structure illustrates one possible mode of interaction between PFL and the activase in which the glycine loop extends across the surface of the PFL-AE active site.…”

Methane as Fuel for Anaerobic Microorganisms

“…In the proposed catalytic mechanism the glycyl radical (for the EPR signal, see Ref. 82) extracts an H atom from the alkane via a thiyl radical, preferentially from the C2 position, yielding an alkyl radical, which reacts with fumarate, yielding a 2‐alkylsuccinyl radical. This radical reacts with the cysteine residue to 2‐alkylsuccinate, regenerating the thiyl radical, and thus the glycyl radical of the enzyme 83–85 …”

Cluster or periodic, static or dynamic—the challenge of calculating the g tensor of the solid-state glycine radical

“…In the BP86 and PW91 cluster calculations, part of the spin density resides on atoms at the edges of the cluster, reducing the spin density on the C2 carbon to about 70% (instead of roughly 90% in the hybrid cluster calculations and the periodic CP2K calculation). In other computational studies using a cluster approach on similar glycinederived radicals 29,30 no such boundary effects were observed. There, neither the radical nor its surrounding molecules were zwitterionic, as is the case in solid-state glycine.…”

“…In previous works on similar glycyl radicals, 29,30 the composition of a supermolecular cluster model proved to have a far less dramatic influence on the g 2 and g 3 values of the calculated g tensor. But there, the spin density was more distributed over a mostly planar radical structure in which much smaller density shifts occurred as a function of the cluster composition.…”

“…Other computational studies have thoroughly examined the g tensor of similar radicals in glycine-derivatives. 29,30 In these systems, the very small g-anisotropy, delocalized spin density and interaction with the molecular environment proved particularly challenging in the calculation of the g tensor, necessitating primarily a proper choice for the spin-orbit operators and an appropriate treatment of the gauge of the external magnetic vector potential.…”