The active site residue Asn437 in protein R1 of Escherichia coli ribonucleotide reductase makes a H-bond to the 2’-OH group of the substrate. To elucidate its role(s) during catalysis Asn437 was engineered by site-directed mutagenesis to several other side chains (Ala, Ser, Asp, Gln). All mutant proteins were incapable of enzymatic turnover, but promoted rapid protein R2 tyrosyl radical decay in presence of the kcat inhibitor 2’-azido-2’-deoxy-CDP, with similar decay rate constants as the wild type R1. These results show that all N437 mutants can perform 3’-hydrogen abstraction, the first substrate-related step in the reaction mechanism. The most interesting observation was that three of the mutant proteins (N437A/S/D) behaved as suicidal enzymes, by catalyzing a rapid tyrosyl radical decay also in reaction mixtures containing the natural substrate CDP. The suicidal CDP-dependent reaction was interpreted to suggest elimination of the substrate's protonated 2’-OH group in the form of water, a step which has been proposed to drive the 3’-H abstraction step. A furanone-related chromophore was formed in the N437D reaction, indicative of stalling of the reaction mechanism at the reduction step. We conclude that Asn437 is essential for catalysis, but not for 3’-H abstraction. We propose that the suicidal N437A, N347S and N437D mutants also can catalyze the water elimination step, whereas the inert N437Q mutant cannot. Our results suggest that Asn437, apart from hydrogen bonding to the substrate, also participates in the reduction steps of catalysis by class I ribonucleotide reductase.