High-frequency EPR and pulsed Q-band ENDOR studies on the origin of the hydrogen bond in tyrosyl radicals of ribonucleotide reductase R2 proteins from mouse and herpes simplex virus type 1

The g tensor of the tyrosyl radical present in the active R2 protein of ribonucleotide reductase is anisotropic, and the g₁ component is influenced by hydrogen bonding to the oxygen of the tyrosyl ring. We have studied the tyrosyl radical in the R2 protein of Escherichia coli, mouse, and herpes simplex virus type 1 (HSV1) with high-frequency EPR and pulsed ENDOR after reconstitution in D₂O. From the high-frequency EPR measurements the g tensor of the radical in HSV1 RNR R2 was found to be identical to that in mouse R2, indicating he presence of a hydrogen bond to the phenolix oxygen in both cases, and in contrast to that in E. coli R2. The pulsed ENDOR spectra confirmed the absence of an exchangeable proton near the tyrosyl radical in E. coli R2. For mouse and HSV1 R2 clear ENDOR signal of exchanged deuterium was found with a hyperfine splitting of -0.53 MHz (mouse) and -0.56 MHz (HSV1). This was interpreted as a proton at a distance of 1.89 Å (mouse) and 1.86 Å (HSV1) from the phenolic oxygen with an orientation, derived from simulations, in the plane of the tyrosyl ring. The most likely origin of this proton is the water ligand at Fe1. This is in contrast with photosystem II where the hydrogen bonding to the radical Y(D)* was formed by a nearby histidine. The presence of the hydrogen bond to the tyrosyl radical may be related to the faster spin-lattice relaxation for the mouse and HSV1 radical compared to that for the E. coli radical, as measured before by Galli et al. [J. Am. Chem. Soc. 1995, 117, 740-746]. It seems therefore likely that the distance between the tyrosyl radical and the iron-oxygen cluster in mouse and HSV1 R2 proteins is shorter compared to that in E. coli R2. Since the tyrosyl radicals in the HSV1 and mouse R2 proteins are much more accessible to the solvent, the hydrogen bond may play useful role in stabilizing the tyrosyl radical.