Abstract
In the reduction of 3-methylriboflavin (3) by dithiothreitol (DTT) and dithioerythritol (DTE) at constant pH, a change in rate-determining step was observed with increasing [buffer] from one which involves buffer catalysis to another which does not. The buffer catalyzed step followed the rate law (feacl[HSS‘] + fcac2[SS2']) [HA] [FI], while the associated solvent terms obeyed (fca0[HSSH] + /cal[HSS“] + fca2[SS2~])[Fl], The Bronsted plot for fcacl gave a = 0.58, and all general acids including hydronium ion and water lay on a single straight Une. The step which is not buffer catalyzed obeyed fcbl[HSS“] [FI]. The corresponding reduction of 3 by mercaptoethanol (ME) obeyed (fcm0[RS~] + fcml[RSH] + &mm[RS~] [RSH])[F1] and showed no buffer catalysis. The first-order terms were small compared to the second-order term, and their cause is unknown. The second-order term in the monothiol reaction and the results from the dithiol reaction establish that the redox reaction between flavin and thiols proceeds entirely or very largely by way of a covalent adduct which then breaks down to product disulfide and reduced flavin. It is argued that buffer catalysis must occur as general-acid catalysis at N(5) in a step other than breakdown. Mechanisms thus ruled out include hydride transfer, general-base-catalyzed thiol deprotonation in attack, general-acid catalysis at N(l), and attack by thiolate at C(la), C(2), C(4), N(5), C(6), and C(8). Reduced 3 is oxidized by bis(2,2'-dithio-4,4'-dinitrobenzoic acid) (DTNB) and obeyed the rate law (fcrt[FlH2] + Zcrl[FlH"])[DTNB]. The rate constant for reaction of hydrazine with DTNB was more than 104 times smaller than kT] in spite of the greater basicity of hydrazine relative to N(l) of reduced flavin. Mechanisms involving adduct formation at N(l) are thus ruled out. Calculations show that mechanisms involving thiol and flavin radicals are too slow to account for the rate of the overall reaction. A mechanism fully consistent with the data is attack of thiolate at C(4a) with general-acid catalysis at N(5) followed by breakdown of the C(4a) adduct by displacement of reduced flavin anion upon attack of thiolate on sulfur.