FIGURE 6.
Kinetics of anaerobic flavin reduction in WT and K842E nNOSred by excess NADPH (600 nm). Stopped-flow traces were collected at 600 nm after rapidly mixing 7–8 μm CaM-free oxidized WT nNOSred or K842E nNOSred (A) and CaM-bound oxidized WT nNOSred or K842E nNOSred (B) with a 10-fold excess of NADPH at 10 °C as described under “Experimental Procedures.” Inset, plots obtained from collecting traces for longer time frames. Rate constants obtained by fitting the absorbance increase and decrease to single exponential functions are reported in Table 4. The calculated best fit curves are plotted as solid lines. Final concentrations after mixing were as follows: nNOSred enzymes, 3.5–4.0 μm; NADPH, 35–40 μm.