Figure 6.

The Michaelis complex intermediate of 3-methyl-2-cyclohexenone and reduced OYE1-T37A. The reduced enzyme was mixed with the substrate (final concentrations 12 μM and 200 μM, respectively), and the dead time absorbances (3 ms) at various wavelengths (open circles) are shown and proposed to be the spectrum of the Michaelis complex. The spectra of the reduced (open squares) and oxidized (open triangles) mutant enzyme, scaled to the same concentration, are also shown. (Insert) The absorbance change at 452 nm during the dead time of the stopped-flow experiments is plotted against the final concentration of the 3-methyl-2-cyclohexenone.