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Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5- di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C- terminal tail of the radical- generating β subunit to render a chimeric RNR. Inspired by a recent cryo- EM structure, a [Re] photooxidant is located adjacent to Y356[β], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism- based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any β metallocofactor highlights the adaptability of the 10- stranded αβ barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α 2 β 2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β 2 , nucleotide reduction in α 2 . Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y 356 (β) and Y 731 (α) across the α/β interface. Using 2,3,5-F 3 Y 122 -β 2 with 3,5-F 2 Y 731 -α 2 , GDP (substrate) and TTP (allosteric effector), a Y 356 • intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz 19 F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y 356 • and the 19 F nuclei of 3,5-F 2 Y 731 in this RNR mutant. Similar experiments with the double mutant E 52 Q/F 3 Y 122 -β 2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F 2 Y 731 . Remarkably, one conformation is consistent with 3,5-F 2 Y 731 within the H-bond distance to Y 356 • , whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y 356 and Y 731 . The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.