Flavin-dependent substrate photo-oxidation as a chemical model of dehydrogenase action

Abstract

As a model of flavin-dependent biological dehydrogenation, flavin-sensitized photodehydrogenation and photodecarboxylation were studied by variation of substrate, flavin, pH and solvent. Evidence for the following rules is given. (1) When the reactive site of a photosubstrate is an alpha-carbon atom of the type CH-CO2-, decarboxylation is preferred over dehydrogenation, whereas the reverse is true for the neutral CH-CO2H. (2) Consequently these reactions do not exhibit a measurable isotope effect with C2H-CO2-, in contrast with the findings by Penzer, Radda, Taylor & Taylor [(1970) Vitam. Horm. (N.Y.) 28, 441--466], which could not be reproduced. When the substate does not contain a carboxylate group, isotope effects occur, in verification of previous reports, e.g. for benzyl alcohol C6H5-C2H20H. (3) The mechanism of flavin-sensitized substrate photodecarboxylation is assumed to consist in a primary carbanion fixation at the flavin nucleus (position 4a, 5 or 8) with concomitant liberation of CO2. This step is followed by rapid fragmentation of the adduct CH-Fl-red., provided that the substrate contains a functional and electron-donating group X, e.g. X = OH, OCH3 or NH2 (but not NH3+ !) in X CH-CO2-. (4) The minimal requirement for flavin-sensitized C-H dehydrogenation is the presence of a hydroxyl group. For example, methanol as substrate and solvent is dehydrogenated at pH sufficiently alkaline for detection of the presence of the active species CH3O-, whereas at more acidic pH substrate dehydrogenation is competing with flavin autophotolysis, which depends on the substituents in the flavin nucleus.

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

“…Figure 11 shows the pH dependence of photoreduction of aqueous 3-me-LF1 by EDTA. This result corresponds to that obtained by Haas and Hemmerich [13] as measured in water/methanol (vol/vol=l:l). The pK's of EDTA are marked by arrows indicating that they are probably responsible for the observed pH dependence.…”

“…The pK's of EDTA are marked by arrows indicating that they are probably responsible for the observed pH dependence. As previously observed [13], the substrate pK gives rise to an increase in the photore- by the arrow duction rate with increasing pH, whereas a flavinphoto-pK leads to a decrease. In the present case, however, the latter is probably overcompensated for by the greater effect of the corresponding pK's of EDTA.…”

“…The dependence of flavin redox reactions under isotropic conditions has been extensively studied by Haas [12] and Haas and Hemmerich [13] in this laboratory. Figure 11 shows the pH dependence of photoreduction of aqueous 3-me-LF1 by EDTA.…”

On the redox reactions and accessibility of amphiphilic flavins in artificial membrane vesicles

“…Thus, Heelis et al are in disagreement with the very bulk of flavin literature if they claim that they observed the same results under aerobic and anaerobic conditions. Finally, Heelis et al did not distinguish between the two fundamentally different photoreactions termed "photolysis" and "photoreduction" [7,12] and probably observed an overlap of these. It is in this way that we interpret their biphasic kinetics.…”

Distinction of 2e- and le- Reduction Modes of the Flavin Chromophore as Studied by Flash Photolysis

“…The common feature in light-excited and dehydrogenase flavins is apparently the induction of basic character either by an auxiliary basic protein group [30,31] or by the intrinsic basicity generated in the triplet 3Fl,*, [13] which both help to split a substrate KH into R -and H', formally.…”