High-field EPR and ENDOR spectroscopy for proton-coupled electron transfer investigations in E.coli ribonucleotide reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms, thereby providing the essential precursors for DNA synthesis and replication. The E.coli RNR is composed of two homodimeric subunits: alpha2 and beta2. In alpha2 nucleotides are converted to deoxynucleotides and beta2 contains a stable di-iron tyrosyl radical (Y122 ) cofactor. A proton-coupled electron transfer (PCET) mechanism is proposed to occur over a distance of more than 35 Å originating from Y122 and ending up in the generation of a C439 in alpha2 which in turn initiates the nucleotide reduction process. Three conserved redox-active tyrosines (beta2-Y356, alpha2-Y730 and alpha2-Y731) are essential for the radical transfer to Cys439 and are proposed to be connected through hydrogen- bonds during reversible charge migration over both subunits. In order to probe for structural and mechanistic requirements of a PCET event in alpha2, NH2Y has been incorporated through the suppressor tRNA/aminoacyl-tRNAsynthetase methodology at positions 730 and 731, respectively. NH2Y functions hereby as a radical spin probe on the PCET pathway and allows the elucidation of chemical environments through high-field EPR and electron-nuclear double resonance (ENDOR) spectroscopy. In order to characterize the generated 3-aminotyrosyl radical (NH2Y ) in D2O and H2O buffer, multifrequency EPR spectroscopy was first applied on NH2Y730 . The characterization of the electronic and molecular structure of a NH2Y was a crucial step in order to deconvolute the hyperfine couplings of internal protons from external protons which build up a hydrogen-bond network around alpha-NH2Y730 . High-field ENDOR spectroscopy detected two hydrogen-bonds which point into the direction of the next PCET pathway residues and additionally a water molecule which is placed near alpha-NH2Y730 and could have mechanistic functions during the proposed H-atom transfer process. A similar hydrogen-bond network was detected for alpha-NH2Y731 with a water molecule placed near the alpha/beta interface. Recent DFT calculations on the PCET mechanism in alpha2 are hereby in agreement with structural insights obtained from high-field EPR and ENDOR studies.