Michael C. Falk

INTER‐ AND INTRAMOLECULAR EFFECTS OF TYROSYL RESIDUES ON FLAVIN TRIPLETS AND RADICALS AS INVESTIGATED BY FLASH PHOTOLYSIS*

Abstract— Addition of tyrosine or derivatives to aqueous solutions of flavins does not significantly impede either formation of the flavin triplet or the rate of O2 oxidation of the flavin radical generated by reaction of triplet with the phenol. However, the rate of radical decay is decreased.

Excitation energy transfer study of the spatial relationship between the carbonyl and metal cofactors in pig plasma amine oxidase

9-Hydrazinoacridine irreversibly labeled pig plasma amine oxidase by covalent attachment to the active carbonyl cofactor. The visible absorption spectrum of the modified protein displays new absorption bands at 495 and 525 nm. Its emission spectrum exhibited maxima at 415 and 440 nm. In addition, both absorption and emission spectra were insensitive to pH changes between 6 and 10. Phase modulation fluorometry was used to determine fluorescence lifetimes of Zn2+- and Co2+-substituted acridinyl plasma amine oxidase. Energy transfer efficiency was 22%; the distance separating the Co2+ ion (in the copper binding site) and the acridine moiety (the amine substrate binding site) ranges between 11.7 and 14.7 A. This work defines the proximity of the metal and substrate (and hence the carbonyl cofactor) and precludes any direct interaction between Cu2+ and pyrroloquinoline quinone or between Cu2+ and the substrate.

Binding and oxidation-reduction of monoamine oxidase-type 8α-(S-peptidyl)flavins with azotobacter (shethna) flavodoxin

Summary Binding of 8α-(S-peptidyl)flavins, the type of covalently attached flavin within the active site of mitochondrial monoamine oxidase, can occur within the FMN-binding site of Azotobacter flavodoxin. Association results in typical hypochromicity of the visible absorbance and a bathochromic shift. There is a decrease in fluorescence of protein but not flavin and a generation of negative ellipticity in the near-UV band in the CD spectra. Bound peptidyl flavins are reduced with EDTA and light to blue semiquinone complexes. Reduction to the hydroquinone level can be effected with excess free flavin during photoreduction or with excess dithionite. No significant monoamine oxidase activity could be detected. In common with other flavodoxins, the FMN-binding site of Azotobacter flavoprotein affixes the coenzyme within a flexible tryptophanyl-containing pocket that allows the benzenoid edge of the isoalloxazine ring to project toward solvent.