Several 2'-substituted-2'-deoxyribonucleotides are potent inactivators of the enzyme ribonucleotide reductase (RNR), by destroying the essential tyrosyl radical located in subunit R2 or/and covalently alkylating the subunit R1. In the absence of external reductants, the inactivation is achieved by alkylation of subunit R1 by a methylene-3(2H)-furanone. The furanone is generated in solution through degradation of a keto-deoxyribonucleotide intermediate, produced during the inhibitory mechanism of a wide group of 2'-substituted inhibitors, and is easily detected experimentally by UV spectroscopy. Interestingly, the same keto-deoxyribonucleotide is also a proposed intermediate of the normal substrate pathway, but by some unknown reason, it does not dissociate from the active site and does not inactivate the enzyme. Therefore, if the currently accepted mechanism for substrate reduction is correct, there must be some specific reason that makes such a reactive intermediate behave differently, not dissociating from the active site during substrate reduction. In this article, we propose to validate the current substrate mechanism by showing that the keto-deoxyribonucleotide dissociates from the active site only in the case of the inhibitors, and therefore, it corresponds to a viable intermediate in the substrate mechanism. Furthermore, we answer unexplained experimental observations that concern the predomination of the normal reduction mechanism over the abnormal ketone formation in the FdNDP and the release of F-, either in the normal or in the abnormal turnover. For that purpose, we have investigated the interaction between the enzyme and this keto-deoxyribonucleotide generated from the normal substrate and from two widely studied representative inhibitors. A model containing 140 atoms was used to represent the desired structures. The results allowed us to conclude that the solvation free energy of the 2'-substituents, its influence inside the active site, and the charge transfer mechanism from a protein side chain to solution are the thermodynamic driving forces for the intermediate dissociation and subsequent RNR inhibition. Such charge transfer cannot be accomplished by the natural substrate, preventing its dissociation. These results elucidate a paradox which has been unexplained for more than 20 years and further validates both the proposed substrate and inhibition chemical mechanisms.