Table 2.
Optical spectral parameters and heme stoichiometry of oxidized (ox) and dithionite-reduced (red) states of wild type and mutant cyt b561a.
| WT | N78K | H88Q | H88Y | H92Q | H92Y | H109Y | H110Q | H110Y | H161Q | H161Y | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| ASoret/hemeb(ox) | 122 (415) | 113 (415) | 110 (411) | 120 (408) | 120 (416) | 110 (416) | 127 (415) | 115 (415) | 110 (415) | 110 (411) | 130 (414) |
| ASoret/hemeb(red) | 187 (427) | 174 (427) | 120 (427) | 130 (425) | 196 (428) | 183 (428) | 197 (427) | 171 (427) | 159 (427) | 130 (427) | 110 (427) |
| Abeta/hemeb(red) | 14 (530) | 14 (530) | 14 (530) | 14 (536) | 15 (530) | 14 (530) | 16 (529) | 14 (529) | 14 (530) | 14 (531) | 16 (530) |
| Aalpha/hemeb(red) | 30 (561) | 30 (561) | 16 (561) | 16 (561) | 31 (562) | 31 (561) | 33 (560) | 29 (561) | 26 (561) | 17 (560) | 17 (560) |
| ASoret/A280(ox) | 3.2 | 3.3 | nde | Nd | 2.9 | 3.0 | 3.1 | 3.3 | 2.8 | nd | Nd |
| ASoret/A280(red) | 4.9 | 5.2 | nd | Nd | 4.7 | 4.8 | 4.7 | 4.9 | 4.4 | nd | Nd |
| A561(red)-A575(ox)c | 54.4 | 48.9 | nd | Nd | 48.1 | 54.1 | 53.3 | 49.7 | 40.5 | nd | Nd |
| Heme/monomerd | 1.94 | 1.91 | 1.3 | 0.9 | 1.78 | 1.98 | 1.87 | 1.98 | 1.85 | 1.3 | 1.6 |
a
Values represent averages from at least two measurements. Purified protein was used except for the His88 and His161 mutants.
b
Extinction coefficient ((mM heme)−1cm−1); heme determined by pyridine hemochrome assay; peak position (nm) in parentheses.
c
Difference extinction coefficient ((mM monomer)−1cm−1)
d
Heme determined by pyridine hemochrome; recombinant protein determined by assay of total protein and densitometry of electrophoretic gels except for His88 and His161 mutants, where recombinant protein was determined by immunoblot.
e
Not determined.