Differences in Proton-Coupled Electron-Transfer Reactions of Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) between Buffered and Unbuffered Aqueous Solutions

The electrochemical reduction mechanisms of flavin mononucleotide (FMN) in buffered aqueous solutions at pH 3–11 and unbuffered aqueous solutions at pH 2–11 were examined in detail using variable-scan-rate cyclic voltammetry (ν = 0.1–20 V s^–1), controlled-potential bulk electrolysis, UV–vis spectroscopy, and rotating-disk-electrode voltammetry. In buffered solutions at pH 3–5, FMN undergoes a two-electron/two-proton (2e^–/2H⁺) reduction to form FMNH₂ at all scan rates. When the buffered pH is increased to 7–9, FMN undergoes a 2e^– reduction to form FMN^2–, which initially undergoes hydrogen bonding with water molecules, followed by protonation to form FMNH^–. At a low voltammetric scan rate of 0.1 V s^–1, the protonation reaction has sufficient time to take place. However, at a higher scan rate of 20 V s^–1, the proton-transfer reaction is outrun, and upon reversal of the scan direction, less of the FMNH^– is available for oxidation, causing its oxidation peak to decrease in magnitude. In unbuffered aqueous solutions, three major voltammetric waves were observed in different pH ranges. At low pH in unbuffered solutions, where [H⁺] ≥ [FMN], (FMN)­H^– undergoes a 2e^–/2H⁺ reduction to form (FMNH₂)­H^– (wave 1), similar to the mechanism in buffered aqueous solutions at low pH. At midrange pH values (unbuffered), where pH ≤ p<i>K</i>_a of the phosphate group and [FMN] ≥ [H⁺], (FMN)­H^– undergoes a 2e^– reduction to form (FMN^2–)­H^– (wave 2), similar to the mechanism in buffered aqueous solutions at high pH. At high pH (unbuffered), where pH ≥ p<i>K</i>_a = 6.2 of the phosphate group, the phosphate group loses its second proton to be fully deprotonated, forming (FMN)^2–, and this species undergoes a 2e^– reduction to form (FMN^2–)^2– (wave 3)