  Flavin reductase catalyzes the reduction of free flavins by NAD(P)H. As
isolated, Escherichia coli flavin reductase does not contain any flavin
prosthetic group but accommodates both the reduced pyridine nucleotide and the
flavin substrate in a ternary complex prior to oxidoreduction. The reduction of
riboflavin by NADPH catalyzed by flavin reductase has been studied by static
and rapid kinetics absorption spectroscopies. Static absorption spectroscopy
experiments revealed that, in the presence of riboflavin and reduced pyridine
nucleotide, flavin reductase stabilizes, although to a small extent, a
charge-transfer complex of NADP+ and reduced riboflavin. In addition, reduction
of riboflavin was found to be essentially irreversible. Rapid kinetics
absorption spectroscopy studies demonstrated the occurrence of two
intermediates with long-wavelength absorption during the catalytic cycle. Such
intermediate species exhibit spectroscopic properties similar to those of
charge-transfer complexes of oxidized flavin and NAD(P)H, and reduced flavin
and NAD(P)+, respectively, which have been identified as intermediates during
the reaction of flavoenzymes of the ferredoxin-NADP+ reductase family. Thus, a
minimal kinetic scheme for the reaction of flavin reductase with NADPH and
riboflavin can be proposed. After formation of the Michaelis complex of flavin
reductase with NADPH and riboflavin, a first intermediate, identified as a
charge-transfer complex of NADPH and riboflavin, is formed. It is followed by a
second charge-transfer intermediate of enzyme-bound NADP+ and reduced
riboflavin. The latter decays, yielding the Michaelis complex of flavin
reductase with NADP+ and reduced riboflavin, which then dissociates to complete
the reaction. These results support the initial hypothesis of a structural
similarity between flavin reductase and the enzymes of the ferredoxin-NADP+
reductase family and extend it at a functional level.
