Table 1.
Rate constants of the absorbance changes during reaction of reduced wild-type and, T312V and Y244F mutant CytcOs with O2 (pH 7.5). 4e and 3e refer to the four and three-electron reduced CytcOs, respectively. Rate constants were typically determined from at least four experiments. The standard error of the mean was ≤10%. pT, proton transfer; eT, electron transfer.
| Observed absorbance changes | ΔA560 decrease ΔA610 increase# (s−1) | ΔA560; ΔA830 increase (s−1) | 1st proton uptake (s−1) | ΔA610 decrease (s−1) | ΔA560 slow decrease (s−1) | 2nd proton uptake (s−1) |
|---|---|---|---|---|---|---|
| Reaction | formation of state P | fractional eT from CuA to heme b | pT to PLS | formation of state F | final oxidation | see Fig 1b |
| WT 4e# | 68000 (15 μs) | 16000 (62 μs) | 17000 (60 μs) | 1300 (0.8 ms) | 900 (1.1 ms) | 1100 (0.9 ms) |
| T312V 4e | 66000 (15 μs) | 13000 (80 μs) | 8300 (120 μs) | 1200 (0.8 ms) | 170 (6 ms) | 170 (6 ms) |
| Y244F 4e | 44000 (22 μs) | 8500 (120 μs) | 7600 (130 μs) | 900 (1.1 ms) | 190 (5.3 ms) | 190 (5.3 ms) |
| WT 3e | 30000 (30 μs) | no¤ | 14000 (70 μs) | 1000 (1 ms) | no | 1000 (1 ms) |
| T312V 3e | 32000 (30 μs) | no | 8300 (120 μs) | 950 (1.0 ms) | no | 290 (3.4 ms) (30%) |
| Y244F 3e | 29000 (34 μs) | no | 6700 (150 μs) | 900 (1.1 ms) | no | 190 (5.3 ms) (30%) |
| T312S 4e& | 33000 (30 μs) | 6000 (170 μs) | 6000 (170 μs) | 30 (33 ms) | 30 (33 ms) | 25 (40 ms) |
| T315V 4e& | 68000 (15 μs) | 13000 (77 μs) | 8500 (120 μs) | ~1000 (~1 ms) | 200 (5 ms) | 200 (5 ms) |
For the four-electron reduced CytcO (the wild-type and both mutant CytcOs) the absorbance decrease at 560 nm (oxidation of heme b) was about a factor of two faster than the increase in absorbance at 610 nm (formation of PR). This is a relatively small dissimilarity in rates and a detailed discussion of any functional differences would require more detailed studies, which is outside of the scope of the present manuscript. As done previously (21) we consider these two events to be synchronous until further studies are done.
no - not observed
The data are from (24).