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, lumitlavin 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[SzO 42-p, where a = 8.6, 100, and 960 M-‘/~ s-r for ferredoxin, metmyoglobin, and lumiflavin j-acetate, respectively. These results support a mechanism involving SO, as the kinetically important reducing species. With cytochome c and plastocyanin, the dependence of kobs on dithionite concentration is described by the equation, kobs = u[S~O~~--]~/~ + b[S2042-], where a and b are 1450 M-r” s-r and 1.5 X lo4 M-l s-r in the case of cytochrome c and 1100 M-Ii2 s-l and 1.35 X lo5 M-l s-l in the case of plastocyanin. These two proteins apparently react with both SO,and S204*-. Analysis of the reaction of cytochrome c with dithionite in the presence of oxygen gave an estimated rate constant of 47 M-l” s-l for the oxygen-dithionite reaction. The rate constant of the reaction of ferricyanide with Sz042is 1.4 X lo5 M-l S-l. Evidence is presented that ferricyanide also reacts rapidly with SO2 . When dithionite reacts with excess ferricytochome c, lumifiavin 3-acetate, or oxygen, the reaction rate is virtually independent of the oxidant concentration and a limiting first order rate constant of 1.7 SF is approached in each case. This rate constant has been assigned to the monomerization