Protein structural changes and tyrosyl radical-mediated electron transfer reactions in ribonucleotide reductase and model compounds

Abstract

Tyrosyl radicals can facilitate proton-coupled electron transfer (PCET) reactions that are linked to catalysis in many biological systems. One such protein system is ribonucleotide reductase (RNR). This enzyme is responsible for the conversion of ribonucleotides to deoxyribonucleotides. The beta2 subunit of class Ia RNRs contains a diiron cluster and a stable tyrosyl radical (Y122*). Reduction of ribonucleotides is dependent on reversible, long-distance PCET reactions involving Y122* located 35 Å from the active site. Protein conformational dynamics are postulated to precede diiron cluster assembly and PCET reactions in RNR. Using UV resonance Raman spectroscopy, we identified structural changes to histidine, tyrosine, and tryptophan residues with metal cluster assembly in beta2. With a reaction-induced infrared spectroscopic technique, local amide bond structural changes, which are associated with the reduction of Y122*, were observed. Moreover, infrared spectroscopy of tyrosine-containing pentapeptide model compounds supported the hypothesis that local amide bonds are perturbed with tyrosyl radical formation. These findings demonstrate the importance of the amino acid primary sequence and amide bonds on tyrosyl radical redox changes. We also investigated the function of a unique tyrosine-histidine cross-link, which is found in the active site of cytochrome c oxidase (CcO). Spectrophotometric titrations of model compounds that mimic the cross-link were consistent with a proton transfer role in CcO. Infrared spectroscopic data support the formation of tyrosyl radicals in these model compounds. Collectively, the effect of the local structure and the corresponding protein dynamics involved in tyrosyl radical-mediated PCET reactions are illustrated in this work.