Figure 2: Reduction of ThnA4ox by NADH.
(a) Spectral evolution of ThnA4 (~2.5 μM) along reaction with NADH (~2.5 μM) as measured by stopped-flow spectroscopy under anaerobic conditions. The thick dashed black line corresponds to the spectrum of ThnA4ox before mixing. Spectra recorded at 0.08064, 0.1626, 0.3264, 0.8179, 1.309, 2.456, 3.931, 5.242, 7.29, 10.07, and 30.23 s after mixing are shown. Directions of the spectral evolutions are indicated by arrows. (b) Evolution of the absorbance at 458 nm (line) and 625 nm (dotted line) and their corresponding global fits (bold lines) to a two-steps model, A → B → C. (c) Spectroscopic properties of the intermediate pre-steady-state species. The inset shows the evolution of the obtained spectral species over the time. Species A, B and C are shown as continuous black thin, black bold and grey bold lines, respectively. (d) Observed kA→B values as a function of NADH (filled squares) and NADPH (open circles) concentrations. Lines represent data fit to Eq. 1. Experimental conditions as in Fig. 1.
