Sulfoxylate Ion (HSO2-), the Hydride Donor in Dithionite-Dependent Reduction of NAD+ Analogues

Article abstract of DOI:10.1021/ja00523a028

At high pH interaction of dithionite with NAD⁺ analogues results in formation of a sulfinate adduct.Its rate of formation is linearly dependent on dithionite concentration.Hence, sulfinate radicals do not appear to be involved in this process.A linear free energy relationship for adduct formation is obtained, the rate of which increases with increasing redox potential of the NAD⁺ analogue.The deproptonated adducts are found to be very stable both thermodynamically (K_d < 10^-7 M) and kinetically (k_off < 10^-4 s^-1).Formation of NADH analogues is therefore not observed at pH > 11.Conversion of adducts, formed from stoichiometric amounts of NAD⁺ analogue and dithionite at high pH, to NADH analogues can be studied by pH jump, stopped-flow spectrophotometry: (1) After protonation of the sulfinate function, formation of oxidized NAD⁺ analogue is observed in a fast initial phase (k for NAD⁺ = 4.62 s^-1), the rate of which increases with decreasing redox potential of nicotinamide. (2) In a much slower, second phase, formation of NADH analogue is observed, which takes more than 20 min to completion.NADH formation can be prevented by adding formaldehyde, which traps the active reducing species. (3) If NAD sulfinate is mixed at pH 5 with an equimolar amount of the high-potential analogue 3-acetylpyridine-NAD⁺ almost quantitative formation of 3-acetylpyridine-NADH is observed with no detectable formation of NADH.These results lead us to propose that sulfoxylate ion (HSO2^-), a hydride donor formed after heterolytic dissociation of the protonated sulfinate adduct, is the active reducing species.Neither the sulfinate adduct itself nor sulfinate radicals appear to be productive in NADH formation.Hence, dithionite appears to be a selective, ambivalent reducing agent.While flavins are deruced by the homolytic dissociation product, sulfinate radical, nicotinamides are reduced by the heterolytic dissociation product, sulfoxylate ion.The factors controlling the nicotinamide pathway are both the high thermodynamic instability of the nicotinamide radical and the high stability of the sulfinate adduct.