On the Existence of Spectrally Distinct Classes of Flavoprotein Semiquinones. A New Method for the Quantitative Production of Flavoprotein Semiquinones^*

Abstract

This work was performed during tenure of Career Development Award GM-K3-31.213.1 Abbreviations used: epr, electron paramagnetic resonance;FMN, riboflavin 5'-phosphate; TPNH, reduced triphosphopyridine nucleotide; FAD, FADH, FADHi, oxidized, semiquinoid, and fully reduced flavin-adenine dinucleotide; TPN+, oxidized triphosphopyridine nucleotide.cals, which are very readily distinguished on the basis of their optical absorption properties. With one enzyme, glucose oxidase, both types of radical are found, the concentrations depending on the pH. These results suggest that the two different spectral species may be due to the neutral and anionic radical forms of the flavin coenzyme prosthetic groups.et al., 1966) and Azotobacter flavoprotein (Beinert, 1965). Again this long wavelength absorption has been found to be well correlated with an epr-detectable free radical, and experimental extinction coefficients in the range 3000-5000 1. mole-1 cm-1 have been found at 570 µ. In the case of two other flavoproteins, d-

Flavin Radical Chelates as Studied by Electron Spin Resonance and Isotopic Substitution

“…The about ten-fold increase in stability constant when going from water to the less polar solvent dimethylformamide indicates that metal flavin radical chelates could have a considerable stability when the flavin is situated in a partly hydrophobic crevice of a protein. The anionic character of the radical ligand is interesting since anionic radicals have been observed in flavoenzymes [28,32].…”

Flavin and 5‐deazaflavin: A chemical evaluation of ‘modified’ flavoproteins with respect to the mechanisms of redox biocatalysis

“…The blue radical species 5-Hill can be further stabilized chemically by alkylation, as shown with the derivatives 5-R61 [49] or biochemically via binding to a suitable apoprotein, as found in the wide-spread group of 'blue radical flavoproteins' [7,9] . In all cases where flavin radicals could be shown to act as catalytically essential intermediates, the radicals are of the blue variety 5-H61, and the stabilization is thought to be brought about by a regiospecific hydrogen bridge pointing from the apoprotein towards N(5) (see below).…”

“…By stabilization through hydrogen bonding to these same centers, it is possible that a similar situation may exist in flavoprotein red radicals. Hence, as mainly disregarded in the earlier literature, red flavoprotein radicals can either be anions Fl-or neutral tautomers of the type l-or 2a-Rl?l, and a clear distinction is only possible in those few cases where a radical pK is seen in the protein-bound state, such as glucose oxidase [7] and lysine monooxygenase [55] . Furthermore, whenever a flavoprotein exhibits a red radical, it appears that the radical state is biocatalytically nonessential, i.e., the red radical cannot be generated by the action of substrates, but only artificially.…”

On the Mechanisms of Photochemical Reductions of FAD and FAD‐Dependent Flavoproteins

“…Also a large excess of benzoate considerably lengthens the time for photoreduction of L-amino acid oxidase a t pH 8. 3 and glucose oxidase at pH 6.…”

“…Recently Massey and Palmer [3] demonstrated that several flavoproteins could be photoreduced with EDTA, but other potential photoreductants were not examined.…”