Table 2.
Steady-state tryptophan fluorescence of FeChTF and His349 FeChTF mutants
| Constructa | λmax (nm) (Fe3+)b | λmax (nm) (apo)c | Fluorescence intensity (counts/s) (Fe3+) | Fluorescence intensity (counts/s) (apo) | Increase Fe3+ → apo pH 5.6 (%)d |
|---|---|---|---|---|---|
| FeChTF | 336 | 336 | 80,000 | 136,000 | 69 ± 5 |
| H349A FeChTF | 335 | 335 | 80,000 | 137,000 | 70 ± 1 |
| H349D FeChTF | 336 | 336 | 81,000 | 103,000 | 31 ± 6 |
| H349K FeChTF | 335 | 334 | 79,000 | 134,000 | 69 ± 0.4 |
| H349L FeChTF | 335 | 336 | 87,000 | 144,000 | 66 ± 0.3 |
| H349W FeChTF | 338 | 337 | 91,000 | 119,000 | 30 ± 2 |
| H349Y FeChTF | 334 | 335 | 85,000 | 130,000 | 51 ± 4 |
a
Spectra were all collected on the same day, allowing direct comparisons of the fluorescence intensities
b
Iron-containing samples were in 100 mM HEPES buffer, pH 7.4
c
Putative apo samples were incubated in 100 mM 2-morpholinoethanesulfonic acid (MES) buffer, pH 5.6, containing 300 mM KCl and 4 mM EDTA for 10–15 min
d
Percent increase is calculated as (100 × [fluorescence intensity(apo) − fluorescence intensity(iron)]/fluorescence intensity(iron)), where fluorescence intensity is measured in counts per second and is an average of data from at least two different experiments