Low-barrier hydrogen-bond powers long-range radical transfer in the metal-free ribonucleotide reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotide (RNA) to deoxyribonucleotide (DNA) building blocks initiated by a long-range (>30 [A]) proton-coupled electron transfer (PCET) by mechanistic principles that remain much debated. By combing multiscale quantum and classical simulations with directed mutagenesis, x-ray crystallography, and vibrational and electron paramagnetic resonance spectroscopy, we elucidate here the molecular principles underlying how metal-free RNRs initiate the long-range PCET process by creating a highly stable DOPA initiator radical. We show that DOPA* is redox-tuned by a low-barrier hydrogen bond (LBHB), with a delocalized proton that provides the catalytic power for the ribonucleotide reduction. We find that the LBHB couples to an extended hydrogen-bonded network, with distant mutations resulting in the loss of radical formation, and providing key molecular insight into the long-range radical transport mechanism in RNRs. On a general level, our findings support the direct involvement of LBHB in protein chemistry and the importance of quantum effects in enzyme catalysis. Significance StatementRibonucleotide reductases (RNRs) are ancient enzymes responsible for the synthesis of deoxyribonucleotides from ribonucleotides. RNRs catalyze this reaction via a long-range proton-coupled electron transfer (PCET) process, involving the formation of a stable protein radical. Yet, despite decades of detailed structural, biochemical, spectroscopic and computational studies, the mechanistic principles of this process remain unclear and much debated. Here, we show that metal-free RNRs power the reduction of RNA building blocks by a highly stable organic DOPA initiator radical, arising from a unique low-barrier hydrogen bonding (LBHB) network that enables the radical transport by strong redox-tuning effects. Our findings reveal mechanistic principles underlying the elusive PCET reactions of metal-free RNRs, and provide evidence for the involvement of quantum effects in enzyme catalysis.