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The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes a ping-pong reaction with NADH and 5,10-methylenetetrahydrofolate (CH 2 -H 4 folate) to produce NAD + and 5-methyltetrahydrofolate (CH 3 -H 4 folate). This work focuses on the function of the invariant, active-site aminoacyl residue Gln183. X-ray structures of the enzyme complexes E red (wild-type)•NADH and E ox (Glu28Gln)•CH 3 -H 4 folate indicate that Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective half-reactions, suggesting roles in binding each substrate. We propose that the polarity of Gln183 may also aid in stabilizing the proposed 5-iminium cation intermediate during catalysis in the oxidative half-reaction with folate. We have prepared mutants Gln183Ala and Gln183Glu, which we hypothesize to have altered charge/polarity and hydrogen bonding properties. We have examined the enzymes by steady-state and stopped-flow kinetics and by measurement of the flavin redox potentials. In the reductive half-reaction, NADH binding affinity and the rate of flavin reduction have not been hindered by either mutation. By contrast, our results support a minor role for Gln183 in the oxidative half-reaction. The Gln183Ala variant exhibited a 6-10 fold lower rate of folate reduction and bound CH 2 -H 4 folate with 7-fold lower affinity, whereas the Gln183Glu mutant displayed catalytic constants within 3-fold of the wild-type enzyme., (Copyright © 2018 Elsevier Inc. All rights reserved.)