Methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr): Protonation state of the ligand and active-site residues

Methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) catalyzes the transfer of the N5-methyl group from N5- methyltetrahydrofolate (CH₃THF) to the cobalt center of a corrinoid/iron-sulfur protein, a reaction similar to that of cobalamin-dependent methionine synthase (MetH). For such a reaction to occur, CH₃THF is expected to be activated by a stereospecific protonation at the N5 position. It has been shown experimentally that binding to MeTr is associated with a pK _a increase and proton uptake. The enzyme could achieve this by binding the unprotonated form of CH₃THF, followed by specific protonation at the correct orientation. Here we have used computational approaches to investigate the protonation state of the ligand and active-site residues in MeTr. First, quantum mechanical (QM) methods with the PCM solvation model were used to predict protonation positions and pK_a values of pterin, folate, and their analogues in an aqueous environment. After a reliable calibration of computational and experimental results was obtained, the effect of the protein environment was then considered. Different protonation states of CH₃THF and activesite aspartic residues (D75 and D160) were investigated using QM calculations of active-site fragment complexes and the perturbed quantum atom (PQA) approach within QM/MM simulations. The final free energy results indicate that the N5 position of the tetrahydropterin ring is the preferred protonation position of CH₃THF when bound to the active site of MeTr, followed by Asp160. We also found that the active-site environment is likely to increase the pK_a of N5 by about 3 units, leading to proton uptake upon CH₃THF binding, as observed experimentally for MeTr. Some implications of the results are discussed for the MetH mechanism.