A New Intermediate in TPNH-Linked Flavoproteins

Abstract

The early studies of Michaelis et. al. (1) clearly indicated that partial reduction of the flavin coenzymes led to the production of colored radical intermediates. Except for the catio-nic radical, which is produced in high yield at very acid pH values (pH<l) the ascription of discrete spectra to the flavin semiquinone was very much hampered by the low radical yield in half reduced flavin-mixtures (2). Further complications arose from the recognition of the existence of charge transfer complexes between reduced and oxidized flavin (2,3,4) with considerable absorption at wavelengths greater than 500 mμ. In fact systematic studies were needed with flavoproteins (5) as well as chemical models (6), (where practically quantitative reduction to the semiquinone can be achieved) in order to show that the neutral flavin semiquinone had a characteristic blue color due to high absorption at 600 my whereas the anionic semiquinone had a characteristic red color, due to the absorption maximum in the region 470–490 my. Figure 1 shows the two semiquinoid forms of glucose oxidase. Except for lipoyl dehydrogenase and glutathione reductase which are complicated by interaction of the flavin with the active center disulfide-dithiol (7,8) all simple flavoproteins so far tested yield either the neutral or anion radical with spectra similar to the two shown in Figure 1. In only two flavoprotein enzymes hitherto studied has the true semiquinoid form been shown unequivocally to be concerned in catalysis (9,10). Instead, with a number of enzymes, the catalytic intermediate appears to be a complex of the flavin and substrate in a partial reduction state, as in the case of D- and L-amino acid oxidases (11,12) or of the two redox active groups of the enzyme (flavin and disulfide) as in the case of lipoyl dehydrogenase (13) and glutathione reductase (8).

Supported by grants from the U.S. Public Health Service, GM 11106 and AM 09313.