FIGURE 2.

Oxidative half-reaction of reductase_TOL with various electron acceptors. Time-dependent spectral changes of 15 μm NADH-reduced reductase_TOL reacting with 30 μm potassium ferricyanide (A) and 8 μm dithionite-reduced reductase_TOL reacting with 20 μm potassium ferricyanide (B). The dashed line represents the spectrum of reduced reductase_TOL, and arrows indicate the direction of absorbance changes. C, time-dependent absorbance change at 450, 600, and 690 nm for the reaction shown in A. D, time-dependent change in absorption at 450 nm when reduced reductase_TOL is mixed with an air-saturated solution. The trace labeled − NAD⁺ shows the reoxidation of dithionite-reduced reductase_TOL, whereas that labeled + NAD⁺ shows the recorded trace for NADH-reduced reductase_TOL where the NAD⁺-reductase_TOL^CT is found in solution. E, absorption spectra of oxidized reductase_TOL (solid black line), oxidized ferredoxin_TOL (solid gray line), NAD⁺-reductase_TOL^CT (dashed black line), and sodium dithionite-reduced ferredoxin_TOL (dashed gray line). F, stopped-flow trace at 498 nm for the reaction of 19.8 μm reductase_TOL with 10 μm ferredoxin_TOL. At this wavelength, the kinetics of the electron transfer to the iron-sulfur cluster of ferredoxin_TOL is observed without significant contributions from the flavin species. The solid line shows a double exponential fit to the data, yielding observed rate constants of 153.8 and 21.4 s⁻¹ and amplitudes of 0.0075 and 0.0056, respectively.