The kinetics of flavine oxidation--reduction. I. Dismutation in nonaqueous solvent.

The dismutation reactions of flavines in dimethylformanide have been investigated using the stopped-flow technique under anaerobic conditions. The ionization constants of fully reduced and oxidized tetraacetylriboflavine were measured spectrophotometrically in buffered dimethylformanide. The dismutation equilibrium of the flavine as a function of pH in dimethylformanide was roughly comparable to that reported in water and allowed the estimation of the pKa value of the flavosemiquinone. The dismutation kinetics of tetraacetylriboflavine in unbuffered dimethylformanide were investigated using the fully oxidized and reduced flavines in their neutral form at constant produce of concentrations and varying the reduction degree. The kinetics at very low reduction ratios (less than3%) were triphasic. The kinetic analysis of the initial and simultaneous formation of the anionic and neutral radicals revealed a second-order reaction. The electron transfer between the oxidized and reduced flavines was not directly coupled with prton exchange. The multiphasic time course of the reaction proceeded primarily from differences in the intrinsic rates of the direct and mixed backward dismutation reactions of the two radical species, and finally from a change in the equilibrium conditions resulting from the accumulation of anionic flavohydroquinone. An acidic-basic negative catalytic effect from the neutral flavohydroquinone appeared progressively as the reduction degree was increased. It was complete at reduction ratios higher than 30%, i.e. under conditions where the radical anion could not be observed at any reaction time. Acids with a pKa value lower than the second one of the flavosemiquinone exhibited a similar catalytic effect. These acidic-basic catalytic effects are associated with changes in the ionic state of labile intermediate dimers formed in the forward as well as in the backward direactions of the dismutation reaction. Such a transient complex revealed by the kinetic analysis could be observed directly by absorption spectroscopy in alkaline-buffered dimethylformanide. Its spectral characteristics, as well as the kinetic effects induced by substitution of the benzenoid part of the flavine, can hardly be taken into account by a quinhydrone-like structure for the intermediate dimers at any pH value. The experimental results favored a more specific interaction, possibly of covalent character, involving the benzenoid part of the isoalloxazine ring.