Methylenetetrahydrofolate reductase from pig liver is a flavoprotein that catalyzes the reduction of N5,10-methylenetetrahydrofolate (CH2-H4folate) to N5-methyltetrahydrofolate (CH3-H4folate) using reducing equivalents supplied by NADPH. In these studies, the physiological cofactor for methylenetetrahydrofolate reductase, flavin adenine dinucleotide, has been replaced by 8-demethyl-8-hydroxy-5-deaza-5-carbaflavin adenine dinucleotide hydroquinone (8-OH-5-deazaFADH2). Using this flavin analogue, the stereochemistry of hydrogen transfer between the deazaflavin hydroquinone and both NADP+ and CH2-H4folate has been determined. Tritium from (5S)-[5-H-3]-8-OH-5-deazaFADH2 is incorporated quantitatively into (4S)-[4-H-3]NADPH or into the methyl group of CH3-H4folate. This is the first example of a flavoprotein that reacts with pyridine nucleotides on the si, rather than the re, face of 8-OH-5-deazaFADH2. The re and si faces of the flavin isoalloxizine are defined relative to C5 of the oxidized flavin. The observation that both NADP+ and CH2-H4folate react at the same face of the enzyme-bound deazaflavin is consistent with the bi-bi ping-pong kinetics previously observed with this enzyme [Matthews, R. G.; Haywood, B. J. Biochemistry 1979, 18, 48451 and supports a mechanism for the NADPH-dependent reduction of CH2-H4folate in which NADP+ dissociates before CH2-H4folate binds the reduced enzyme. In the presence of 8-OH-5-deazaFAD, methylenetetrahydrofolate reductase apoenzyme has a k(cat) value for the NADPH-CH2-H4folate oxidoreductase reaction that is 52% of that observed for the apoenzyme assayed in the presence of FAD. These observations suggest hydride transfer mechanisms for the reduction of enzyme-bound flavin by NADPH and its reoxidation by CH2-H4folate.
