Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism that binds flavin adenine dinucleotide (FAD) and uses NAD(P)H to catalyze the conversion of 5,10‐methylenetetrahydrofolate to 5‐methyltetrahydrofolate by a ping‐pong Bi‐Bi mechanism. The x‐ray crystal structure of the 5‐methyltetrahydrofolate and FAD ‐bound E. coli enzyme has revealed a hydrogen bonding network that involves amino acid residues Ser26, His273, Glu28, a structural water molecule, and the N10 of the folate. Based on previous studies that confirmed the importance of Glu28 in the catalysis of the folate half‐reaction, we hypothesized a proton relay mechanism that transports a proton from the aqueous solvent to the N10 of the folate through this hydrogen bonding network and catalyzes the formation of the putative 5‐iminium cation folate intermediate. In our study, we employed steady‐state oxidoreductase assays using menadione as an artificial electron receptor and measured kinetic parameters of the wild‐type E. coli enzyme and two mutants, His273Gln and His273Ala MTHFRs. We found that in the methyltetrahydrofolate‐menadione assay, where the folate half‐reaction is rate‐limiting, the kcat of the His273Ala mutant (0.047 ± 0.002 s−1) is more than 10‐fold lower than the wild‐type enzyme (kcat = 0.54 ± 0.01 s−1). His273Gln mutant showed similar activity to the wild‐type enzyme, but strong competitive substrate inhibition was observed. In the NADH‐menadione assay, both His273Ala and His273Gln showed insignificant change in kcat (1.2 – 1.3‐fold) and in Km (1.7 – 2‐fold). Together, these results suggest that the ability of the His273 residue to form hydrogen bonds is important in the catalysis of the folate half‐reaction and support the hypothetical proton relay mechanism. In addition, we report our use of Liquid Chromatography‐Mass Spectrometry (LC‐MS) to quantify the product in the folate‐menadione assay more accurately.Support or Funding InformationGrinnell College Department of ChemistryHydrogen‐bonding network in the X‐ray structure of E28Q MTHFRFigure 1v/E vs. [substrate] plots obtained from methyltetrahydrofolate‐menadione oxidoreductase assays at 4C.Figure 2