Figure 5.

Determination of the mid point potential at pH 7.0 of the R181M enzyme form and the effect of pyruvate concentration on the apparent E_m value. The concentrations of oxidized (E_ox) and reduced (E_red) enzyme forms were determined from the spectra at equilibrium on mixing enzyme under anaerobic conditions in a Hi-Tech model SF-61 stopped flow spectrophotometer at fixed pyruvate concentrations of 2.5 mM (⧫), 10 mM (▾), 20 mM (▴), and 40 mM (●). The E_m apparent values were determined from the intercept values of log (E_ox/E_red) and are shown in the Inset. The line through the points of the inset is a theoretical one for an E_m value of −140 mV in the absence of pyruvate (see text for details).