TABLE 3.
Rates of anaerobic flavin reduction of nNOSred proteins by excess NADPH at 457 nm
Reductions were carried out in the stopped-flow instrument at 10 °C with a 10-fold excess of NADPH while monitoring the absorbance changes at 457 nm. Data were fit to a triple exponential function as described under “Experimental Procedures.”
| Enzyme | Conditions | Dead timea | k1b | k2b | k3b |
|---|---|---|---|---|---|
| % | s−1 | s−1 | s−1 | ||
| Wild type | −CaM | 30 | 63 ± 7.2 (24%) | 6 ± 0.28 (31%) | 0.86 ± 0.022 (15%) |
| +CaM | 21 | 146 ± 3.7 (21%) | 15 ± 0.6 (33%) | 2.2 ± 0.16 (25%) | |
| K842E | −CaM | 31 | 157 ± 5.7 (33%) | 23 ± 2 (10%) | 3.8 ± 0.3 (26%) |
| +CaM | 25 | 161 ± 8 (42%) | 28 ± 2.3 (14%) | 7.6 ± 0.4 (19%) |
a Percentage of the total absorbance change occurring in the instrument dead time.
b Individual rate constants are reported as follows: calculated rate (s−1) and percentage of the total absorbance change for this process in parentheses.