TABLE 1.
Summary of the rate constants for the reactions of H120N·FMNH−, H120Q·FMNH−, H120Y·FMNH−, H120D·FMNH−, H120E·FMNH−, H120K·FMNH−, S146A·FMNH−, and S146C·FMNH− with oxygen in the absence and presence of HPA
Rate constants are as described in Figs. 3 and 4 and from stopped-flow and rapid-quench data in Figs. 2 and 6 and supplemental Figs. 1–5 and 8–9.
| Enzymes | Rate constant of C4a-hydroperoxy-FMN formation (k1) | Rate constant of H2O2 elimination from C4a-hydroperoxy-FMN (k2) | Rate constant of C4a-hydroperoxy-FMN·HPA formation (k3) | Rate constant of hydroxylation (k4) | Rate constant of C4a-hydroxy-FMN dehydration (k5) |
|---|---|---|---|---|---|
| m−1s−1 | s−1 | m−1s−1 | s−1 | s−1 | |
| C2 wild type | 1.1 × 106a | 3 × 10−3a | 4.8 × 104a | 16 a | 0.35 a |
| H120 variants | |||||
| H120N | 6.2 ± 0.3 × 105 | 1.9 ± 0.2 × 10−2 | |||
| 6.7 ± 0.2 × 105b | 1.2 ± 0.1 × 10−2b | ||||
| H120Q | 7.6 ± 0.2 × 105 | 7.0 ± 0.1 × 10−3 | |||
| H120Y | 5.8 ± 0.1 × 105 | 4.0 ± 0.1 × 10−3 | |||
| H120D | 8.4 ± 0.3 × 105 | 3.3 ± 0.6 × 10−2 | |||
| 8.4 ± 0.3 × 105b | 3.4 ± 0.2 × 10−2b | ||||
| H120E | 5.6 ± 0.1 × 105 | 2.1 ± 0.1 × 10−2 | |||
| H120K | 6.3 ± 0.2 × 105 | 5.2 ± 0.1 × 10−1 | 4.6 ± 0.2 × 104c | 5.7 ± 0.6 | 0.75 ± 0.01 |
| S146 variants | |||||
| S146A | 3.1 ± 0.1 × 105 | 2.0 ± 0.3 × 10−3 | 2.2 ± 0.1 × 104 | 2.6 ± 0.1 | 0.031 ± 0.001 |
| S146C | 1.1 ± 0.1 × 106 | 8.0 ± 0.2 × 10−3 | 6.2 ± 0.3 × 105 | 0.35 ± 0.05 | >0.35 |
a Rate constants of the wild-type enzyme at pH 7.0, 4 °C from (15).
b Rate constants of the reactions of H120N·FMNH− and H120D·FMNH− with oxygen in the presence of HPA. No hydroxylation occurs in these reactions.
c Rate constant of C4a-hydroperoxy-FMN formation in the reaction of H120K in the presence of HPA was taken from the reaction with 0.13 mm oxygen (supplemental Fig. 6).