Class I ribonucleotide reductase revisited: the effect of removing a proton on Glu441

The substrate mechanism of class I ribonucleotide reductase has been revisited using the hybrid density functional B3LYP method. The molecular model used is based on the X-ray structure and includes all the residues of the R1 subunit commonly considered in the RNR substrate conversion scheme: Cys439 initiating the reaction as a thiyl radical, the redox-active cysteines Cys225 and Cys462, and the catalytically important Glu441 and Asn437. In contrast to previous theoretical studies of the overall mechanism, Glu441 is added as an anion. All relevant transition states have been optimized, including one where an electron is transferred 8 A from the disulfide to the substrate simultaneously with a proton transfer from Glu441. The calculated barrier for this step is 19.1 kcal/mol, which can be compared to the rate-limiting barrier indicated by experiments of about 17 kcal/mol. Even though the calculated barrier is somewhat higher than the experimental limit, the discrepancy is within the normal error bounds of B3LYP. The suggestion from the present modeling study is thus that a protonated Glu441 does not need to be present at the active site from the beginning of the catalytic cycle. However, the previously suggested mechanism with an initial protonation of Glu441 cannot be ruled out, because even with the cost added for protonation of Glu441 with a typical pK(a) of 4, the barrier for that mechanism is lower than the one obtained for the present mechanism. The results are compared to experimental results and suggestions.