TABLE 3.
Effect of CaM and NADPH on FMN shielding in fully reduced NOSr proteins An excess of each pre-reduced NOSr protein in the presence or absence of NADPH and CaM was mixed with cytochrome c in a stopped-flow instrument under anaerobic conditions at 10 °C. The molar ratio of NOSr enzyme to cytochrome c was 5:1. The observed rates of absorbance increase at 550 nm are reported as the mean ± S.D. of 5–6 single mixing experiments and are representative of at least two different enzyme preparations. The values in parentheses are the relative percentage of absorbance change with respect to the full reduction of 1.5 μm cytochrome c used in the reaction. ND is not detected.
|
Enzyme
|
Observed rate
|
||
|---|---|---|---|
| –CaM/–NADPH | –CaM/+NADPH | +CaM/+NADPH | |
| s–1 | |||
| eNOSr | |||
| k1 | 52.9 ± 1.8 (54%) | 60.9 ± 3.4 (81%) | 72.5 ± 3.9 (100%)a |
| k2 | 7.1 ± 0.2 (46%) | 16.3 ± 1.2 (19%) | ND |
| nNOSr | 23 ± 1.2 | 5.8 ± 0.5 | 41 ± 0.5 |
For eNOSr, the total absorption change measured after 0.5 s corresponded to 85% of the expected calculated absorbance change at 550 nm for cytochrome c