Table 2.
Effect of the mutations in the ef loop of cyt b on the kinetics of electron transfer within R. sphaeroides cyt bc1.
| Cyt bc1 Mutanta | Specific Activityb | k1c (s−1) | Famoxadone k1 (s−1) | k2d(s−1) | Δ CAe(Å) |
|---|---|---|---|---|---|
| WT | 2.35 | 60,000 | 5,400 | 2,300 | |
| H276A (D252) | 1.56 | 42,000 | 2,800 | 2,200 | 3.3 |
| P277A (P253) | 1.10 | 35,000 | 4,500 | 1,700 | 3.0 |
| D278A (D254) | 1.43 | 26,000 | 2,300 | 2,300 | 2.9 |
| D278N | 1.88 | 35,000 | 2,500 | 2,400 | |
| D278E | 1.60 | 51,000 | 6,700 | 2,900 | |
| D278I | 2.41 | 38,000 | 4,300 | 2,000 | |
| D278H | 1.49 | 36,000 | 4,100 | 1,500 | |
| N279A (N255) | 2.43 | 37,000 | 4,300 | 1,900 | 1.5 |
| Y280A (Y256) | 1.34 | 7,900 | 3,200 | 2,800 | 0.5 |
| L286A (L262) | 0.78 | 33,000 | 35,000 | 740 | |
| L286E | 0.88 | 17,000 | 12,000 | 1,900 | |
| L286I | 2.40 | 18,000 | 2,900 | 2,300 | |
| S287A (N263) | 2.16 | 55,000 | 5,700 | 1,000 | 0.7 |
| A290S (P266) | 2.65 | 49,000 | 5,600 | 1,900 | 0.9 |
| H291A (H267) | 2.54 | 51,000 | 4,100 | 2,000 | 1.4 |
| I292A (I268) | 0.81 | 4,400 | 3,000 | 350 | 1.4 |
| I292L | 1.30 | 10,000 | 4,700 | 1,500 | |
| I292M | 0.92 | 6,200 | 2,800 | 1,700 | |
| I292V | 0.92 | 33,000 | 3,500 | 1,300 | |
| I292E | 0.12 | - | - | - | |
| I292R | 0.56 | 10,000 | 2,500 | 1,700 |
The corresponding residues of bovine cyt b are given in parenthesis.
Specific activity is expressed as micromole of cyt c reduced/min/nmol cyt b at 25 °C using chromatophores. The error limits are ± 15%.
The rate constant k1 for electron transfer from [2Fe2S] to cyt c1 was measured at 552 nm in a solution containing 5 μM cyt bc1, 20 μM Ru2D, 5 mM [Co(NH3)5Cl]2+, in 20 mM sodium borate, pH 9.0, 0.01% dodecylmaltoside. The cyt bc1 was treated with 10 μM QoC10BrH2, 1 mM succinate, and 50 nM SCR to completely reduce [2Fe2S] and cyt c1, and reduce cyt bH by about 30%. Famoxadone (30 μM) was added where indicated. The error limits are ± 20%.
The rate constant k2 for electron transfer from QH2 to [2Fe2S] was measured from the rate of cyt bH reduction at 561−569 nm under the conditions described for k1 above. The error limits are ± 20%. Addition of famoxadone eliminated that 561−569 nm transients for all of the mutants, indicating that famoxadone bound to all the mutants in the Qo pocket.
ΔCA is the displacement of the α carbon of the indicated residue in bovine cyt bc1 induced by famoxadone binding (31).