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DITHIONITE TREATMENT OF FLAVINS: SPECTRAL EVIDENCE FOR COVALENT ADDUCT FORMATION and EFFECT ON in vitro BACTERIAL BIOLUMINESCENCE

Abstract— Intrigued by the apparent requirement of dithionite for FMN reduction (as opposed to photoreduction or catalytic hydrogenation) in the H2O2‐initiated bacterial bioluminescence reaction, we chose 5‐ethyl‐3‐methyllumiflavinium cation I as a mode) to investigate possible flavin adduct formation by treatment with dithionite or (bi)sulfite. In the range of pH5–8, the reaction of dithionite with 5‐ethyl‐3‐methylIumiflavinium cation, which is in equilibrium with the 5‐ethyl‐4a‐hydroxy‐3‐methyl‐4a, 5‐dihydrolumiflavin pseudobase II (X = OH), is not limited to the formation of flavo‐semiquinone and dihydroflavin following two one‐electron steps. Several parallel and sequential reactions may take place involving the intermediacy of covalent flavin adducts. Addition of (bi)sulfite gave a 4a‐sulfiteflavin adduct II (X = SO3). Consistent with the S2O4 ⇄ 2 SO2 equilibrium, the reaction of dithionite and II (X = OH; SO3) gave rise to two flavin adducts in competitive nucleophilic displacements: a 4a‐sulfoxylate‐flavin radical (II, X = SO2) and a 4a‐dithioniteflavin adduct (II, X = S2O4), respectively. On increasing the (S2O4, SO2)/f1avin ratio under N2, the formation of the 4a‐sulfoxylate‐flavin radical became predominant. The II (X = SO2) so formed was in equilibrium with the flavosemiquinone and bisulfite and can be trapped by reacting with hydroxylamine. In the initial presence of oxygen, II (X = SO2) was highly reactive toward O2, giving a fast oxidation to II (X = SO3) and effectively suppressing the formation of the flavosemiquinone. If the oxygen is subsequently removed, the II (X = SO3) so formed then reacts with dithionite leading to quantitative accumulation of II (X = S2O4). Slow, spontaneous decompositions of the 4a‐dithioniteflavin adduct indicated the occurrence of alternative pathways, probably proceeding via a 4a‐sulfoxylateflavin anion (II, X = SO2) and II (X = SO2) as intermediates. The implications of these findings for the reactions of (bi)sulfite and dithionite with (dihydro)flavodoxins and for the role of dithionite in the H2O2 induced bacterial bioluminescence are discussed.