The Kinetics and Mechanism of Reduction of Electron Transfer Proteins and Other Compounds of Biological Interest by Dithionite

The kinetics of the reduction by dithionite of several biochemically interesting substances has been investigated under anaerobic conditions by stopped flow spectrophotometry at pH 8.0 and 25°. Solutions of sodium dithionite were prepared and standardized by titration against lumiflavin 3-acetate in the absence of oxygen. Under pseudo-first order conditions (dithionite in excess) the reactions of spinach ferredoxin, horse metmyoglobin, lumiflavin 3-acetate, horse heart ferricytochrome c, and spinach plastocyanin conform to first order kinetics for several half-lives. Dependence of the observed pseudo-first order rate constant (kobs) on the dithionite concentration is described by the equation, kobs = a[S2O42-]½, where a = 8.6, 100, and 960 m-½s-1 for ferredoxin, metmyoglobin, and lumiflavin 3-acetate, respectively. These results support a mechanism involving SO2- as the kinetically important reducing species. With cytochome c and plastocyanin, the dependence of kobs on dithionite concentration is described by the equation, kobs = a[S2O42-]½ + b[S2O42-], where a and b are 1450 m-½s-1 and 1.5 x 104m-1s-1 in the case of cytochrome c and 1100 m-½s-1 and 1.35 x 105m-1s-1 in the case of plastocyanin. These two proteins apparently react with both SO2- and S2O42-. Analysis of the reaction of cytochrome c with dithionite in the presence of oxygen gave an estimated rate constant of 47 m-½s-1 for the oxygen-dithionite reaction. The rate constant of the reaction of ferricyanide with S2O42- is 1.4 x 105m-1s-1. Evidence is presented that ferricyanide also reacts rapidly with SO2-. When dithionite reacts with excess ferricytochome c, lumiflavin 3-acetate, or oxygen, the reaction rate is virtually independent of the oxidant concentration and a limiting first order rate constant of 1.7 s-1 is approached in each case. This rate constant has been assigned to the monomerization rate of S2O42-, and its temperature dependence gives an activation energy of 24.1 Cal per mole for the dissociation of S2O42-. Determination of the equilibrium constant (K) for the reaction S2O42- ⇌ 2SO2- by electron paramagnetic resonance spectroscopy has given a value of 1.4 ± 0.4 x 10-9m. Dividing the above listed values of a by K½ gives the absolute rate constants for the reactions between SO2- and oxidants.