Abstract
In the reduction of 3-methylriboflavin (3) by dithiothreitol (DTT) a second change in rate-determining step was observed at low [buffer] when [DTT] was low (5-mM range). The third sequential step thus revealed was buffer catalyzed, followed the rate law, fcdc0[B][HSSH][Fl], and is attributed to general-base (B)-catalyzed deprotonation of the -SH group of the C(4a) adduct between DTT and flavin. The Bronsted plot in the case of 3 for deprotonation of adduct -SH resembles that for simple proton transfer from thiols, except that the rate constants of the latter are larger by about 105. This factor of 10s is attributed to the unfavorable equilibrium of adduct formation, KAii « 10~5 M"1. DTT monoanion was an effective general base in deprotonation of the adduct and exhibited the rate law ^[HSS-] [HSSH] [FI], A comparison of the deprotonation rate constants for 3 with those for 7-chlororiboflavin (4) and 7,8-dichlororiboflavin (6) gave ÁTAdd = 2.1 X 10"4 and 3.4 X 10"3 M"1 for 4 and 6, respectively. Difference spectra between 6 and 6 plus mercaptoethanol under conditions precluding net flavin reduction showed, in addition to solvent perturbations, the disappearance of a small fraction of 6 and the concomitant appearance of a new component with „, = 370 nm (c = 15 mM"1 cm"1) by assuming the component to be formed stoi- chiometrically from 6. KAii = 3.4 X 10"3 M"1 agrees well with the value determined kinetically. Flavins 3 and 4 failed, as expected, to give spectrophotometric evidence for adduct formation. The difference spectrum showing the 370-nm component is similar to that of a form of lipoamide dehydrogenase ( „, = 387 nm (e = 8.7 mM"1 cm"1)) which has been attributed to an adduct between flavin and an active site thiol. Implications of our findings to the mechanism of this and analogous enzymes are discussed.