Photoreduction of flavoproteins and other biological compounds catalyzed by deazaflavins. Appendix: photochemical formation of deazaflavin dimers

Abstract

Deazaflavins have been found to act as potent catalysts in the photoreduction of flavoproteins in the presence of EDTA and other "photosubstrates". In distinction to the catalysis brought about by normal flavins which involves dark reaction of the photoreduced flavin catalyst, the mechanism of the catalysis by deazaflavins has been shown to involve unstable, strongly reducing radicals which are generated by photolysis of a preformed covalent dimer. By this new method it is possible to reduce not only flavoproteins but a variety of other redox proteins, including heme proteins and iron-sulfur proteins. By virtue of its great catalytic efficiency, it is possible to employ concentrations of deazaflavin sufficiently low as not to interfere with the spectral evaluation of the reduced proteins obtained.

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

“…10) supports this expectation as the reciprocal plots for the initial velocity data collected with varied concentrations of F 420 H 2 (5-60 M) and Mj-Trx1 at two fixed concentrations (2 and 15 M) produced two parallel lines. Because there were indications of complications arising from inhibition of the enzyme by F 420 H 2 and the generation of single-electron reduced deazaflavin species (37) in the presence of EDTA and visible light used in spectrophotometry, albeit in very minor amounts, the detailed mechanistic studies will be pursued and reported in the future. It should be noted that to avoid such a problem, all redox titrations were performed in the absence of EDTA.…”

“…7B). To avoid the wavelengths where all or most of the participating cofactors exhibit significant absorbance values (29,37), the A 480 and A 540 values (where A is absorbance) were chosen for the determination of the concentrations of FAD and the FAD-thiolate charge transfer species from which similar information on other species could be calculated. Fig.…”

2-Thioriboflavin 5'-Phosphate (2-Thio-FMN) Lactate Osidase

“…For anaerobic experiments Thunberg-type cells or tonometers were used and anaerobiosis was achieved by repeated evacuation and flushing with N, purified over Fieser's solution. Photochemical reactions with 5-deazaflavin as catalyst were done at ice temperature as described previously [18].…”

“…On photoreduction with 5-deazaflavin as catalyst [18] both native lactate oxidase and the iso-FMN enzyme exhibit spectra typical of the flavin anionic semiquinone [7,8]. In both cases the semiquinone is rapidly reoxidized on mixing with 0, and with both forms this oxygen reactivity is dramatically slowed by formation of a complex with pyruvate [7,8].…”

“…8 shows the spectrum of 2-thio-FMN flavodoxin in its three oxidation states. Again, 5-deazaflavin-catalyzed photoreduction was used to generate the semiquinoid and fully reduced forms, as can be done with the native protein [18]. As with the native protein [24], mixing the fully reduced form with O2 results in the very rapid reformation of semiquinone (and 0;) and the very much slower reoxidation of the semiquinone to the oxidized form.…”