The first flavoprotein to be discovered was isolated from yeast by Otto Warburg in 1933 (1) and its flavin identified as riboflavin 5-phosphate (FMN) by Theorell two years later (2). Despite a history of 60 years, the physiological function of this enzyme, commonly referred to as Old Yellow Enzyme, has remained unsolved. It was recognized by Warburg and Theorell that the enzyme was readily reduced by NADPH, and it has become accepted that NADPH is the physiological reductant of the enzyme.

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... er, it was clear that the NADPH-oxygen reductase activity measured in early studies could not be the physiological reaction catalyzed by the enzyme, and that some other naturally occurring compound in yeast must be the physiological electron acceptor. In order to try to determine what the natural substrate might be, we set out nearly thirty years ago to devise a new method of purification of the enzyme, thinking that the rather harsh protein separation methods available to Warburg and Theorell might have resulted in some damage to the enzyme that masked its true function. The new purification procedure, developed by Rowena Matthews (3) immediately gave us hope that we would identify the natural substrate, since the isolated enzyme was bright green in color, and could be converted into the traditional old yellow form by dialysis against dithionite, followed by air reoxidation. It was clear that the green form of the enzyme was due to complex formation of the oxidized enzyme and a "greening factor", which was disrupted on reduction, since the green long wavelength-absorbing form of the enzyme could be reconstituted by addition of the dialysate (after concentration aerobically ) to the yellow form. At this time the occurrence of charge transfer complexes between oxidized flavoproteins and electron-rich Flavins and Flavoproteins 1993