TABLE 2.
The effect of mutation of charged residues on Phormidium PC in the absence of the Zn2+ ion on its interaction with Phormidium cyt f
| Ionic strength (mM)
|
|||||||
|---|---|---|---|---|---|---|---|
| 10
|
100
|
||||||
| Class* | Mutant† | Complexes formed per 1000 trajectories† | % WT | 10−8 × k2 (M−1 s−1)† | Complexes formed per 1000 trajectories† | % WT | 10−8 × k2 (M−1 s−1)† |
| WT | 20.4 ± 0.5 | 100 | 5.9 ± 0.3 | 8.5 ± 0.6 | 100 ± 10 | 2.5 ± 0.4 | |
| I | >200% WT complexes formed at 10 mM ionic strength | ||||||
| D44(42)A | 70.8 ± 0.9 | 403 | 20.2 ± 0.9 | 16.0 ± 0.7 | 188 ± 15 | 4.6 ± 0.5 | |
| D45(43)A | 65.1 ± 1.3 | 370 | 18.7 ± 1.0 | 15.0 ± 0.7 | 176 ± 15 | 4.4 ± 0.4 | |
| D10(8)A | 63.0 ± 1.3 | 358 | 7.6 ± 0.5 | 14.1 ± 0.7 | 165 ± 14 | 4.1 ± 0.4 | |
| E70(68)A | 50.9 ± 0.3 | 289 | 6.2 ± 0.4 | – | – | – | |
| D57(55)A | 50.0 ± 1.1 | 284 | 14.6 ± 0.9 | 12.4 ± 0.6 | 146 ± 13 | 2.6 ± 0.5 | |
| E17A | 39.1 ± 1.2 | 221 | 4.8 ± 0.3 | 12.4 ± 0.6 | 146 ± 12 | 3.6 ± 1.4 | |
| II | 130–200% WT complex formation at 10 mM ionic strength | ||||||
| E54(52)A | 30.9 ± 1.1 | 176 | 3.8 ± 0.3 | 9.3 ± 0.6 | 110 ± 12 | 0.8 ± 0.2 | |
| E1(−3)A | 29.4 ± 0.5 | 167 | 8.9 ± 0.8 | – | – | – | |
| E73(71)A | 29.4 ± 0.8 | 167 | 3.7 ± 0.3 | – | – | – | |
| E104(99)A | 28.1 ± 1.0 | 160 | 3.5 ± 0.3 | – | – | – | |
| III | 100–133% WT complex formation at 10 mM ionic strength | ||||||
| D79(77)A | 23.4 ± 1.0 | 133 | 2.9 ± 0.3 | 9.7 ± 0.6 | 114 ± 11 | 2.7 ± 0.4 | |
| D27(25)A | 22.5 ± 0.7 | 128 | 2.8 ± 0.4 | – | – | – | |
| IV | 40–100% WT complexes formed at 10 mM ionic strength | ||||||
| K58(56)A | 10.1 ± 0.7 | 58 | 1.3 ± 0.3 | 8.0 ± 0.5 | 94 ± 9 | 2.3 ± 0.4 | |
| K53(51)A | 9.9 ± 0.8 | 56 | 2.6 ± 0.5 | 5.8 ± 0.6 | 68 ± 8 | 1.7 ± 0.3 | |
| K30(28)A | 8.5 ± 0.6 | 48 | 1.1 ± 0.3 | – | – | – | |
| V | <40% WT complexes formed at 10 mM ionic strength | ||||||
| K100(95)A | 5.4 ± 0.3 | 31 | 0.8 ± 0.2 | 6.1 ± 0.7 | 76 ± 0.8 | 1.9 ± 0.4 | |
| K46(44)A | 4.6 ± 0.8 | 26 | 1.8 ± 0.4 | 5.5 ± 0.6 | 65 ± 9 | 1.6 ± 0.3 | |
| K6(4)A | 3.9 ± 0.4 | 22 | 0.6 ± 0.2 | 6.4 ± 0.6 | 76 ± 8 | 1.9 ± 0.4 | |
| R93(88)A | 1.3 ± 0.3 | 7 | 0.8 ± 0.3 | 3.0 ± 0.5 | 35 ± 7 | 0.9 ± 0.3 | |
| K35(33)A | 1.1 ± 0.4 | 6 | 0.2 ± 0.2 | 4.1 ± 0.6 | 48 ± 7 | 1.2 ± 0.3 | |
The number of complexes formed in the absence of the electrostatic field (2.2 ± 0.2 complexes per 1000 trajectories) was subtracted from the total number of complexes formed to obtain the number of complexes formed due to electrostatic forces. The mutants were divided into classes based on their effects on complex formation. Class I, significant stimulation of complex formation (i.e., the number of complexes formed was >200% WT at 10 mM ionic strength; class II, 133–200% WT at 10 mM; class III, the number of complexes formed was between 100 and 133% WT at 10 mM; class IV, the number of complexes formed was between 40 and 100% WT at 10 mM; class V, the number of complexes formed was <40% WT at 10 mM ionic strength. The rates of electron transfer were calculated as described in the Methods section and for Table 1. The rate of electron transfer found in the absence of the electrostatic field (0.6 ± 0.2 × 102 M−1 s−1) was subtracted from the observed rates to obtain the rate of electron transfer due to electrostatic forces alone. Five sets of 5000 and 10,000 trajectories were carried out at 10 and 100 mM ionic strength, respectively.
The mutants were made as described in the Methods section. The number of complexes formed and the rates of electron transfer were calculated as described for Table 1 and in the Methods section.