TABLE 2.
Heme reduction and flavin reduction of nNOS and the Lys842 mutants
All reactions were carried out at 10 °C under anaerobic conditions in a stopped-flow spectrophotometer as described under “Experimental Procedures.” k1 and k2 refer to the rates for biphasic reactions. The values for flavin and heme reduction in wild-type and mutant NOSs are the mean ± S.D. of 7–10 individual reactions and are representative of experiments done with two different enzyme preparations. The numbers in parentheses indicate the percentage of absorbance change attributed to the fast phase (k1). ND, not detected.
| Enzyme | CaM | Flavin reduction |
Heme reduction (k) | |
|---|---|---|---|---|
| k1 | k2 | |||
| s−1 | s−1 | |||
| nNOS WT | + | 72 ± 1.5 (40%) | 8.0 ± 0.05 | 3.6 ± 0.01 |
| − | 7.3 ± 0.2 (45%) | 1.5 ± 0.01 | ND | |
| K842A | + | 53 ± 1.3 (52%) | 9.1 ± 0.1 | 2.4 ± 0.01 |
| − | 22 ± 0.6 (55%) | 5.0 ± 0.1 | ND | |
| K842E | + | 78 ± 3.0 (40%) | 10 ± 0.1 | 3.2 ± 0.01 |
| − | 34 ± 0.7 (44%) | 6.7 ± 0.07 | ND | |