Table 2. Properties cNOR variants in predicted proton pathway 2.
Data from T. thermophilus is added for comparison from Ref. [13] and data from E78cF, Q394M and Q398L is from Ref. [7]. New data is highlighted in bold. Conservation and location from Ref. [7].
| Res. In Ps. aer. (conservation) Location | Mutation in P. den. /T. therm. | Spectra | Maximum turnover activity with NO (% of WT activity) a) | CO binding b) τ1 (μs)/ τ2 (μs) (% τ2) c) 0 | O2 b) binding τ (μs) | ETPT d) τ (ms) at pH 7.5 |
|---|---|---|---|---|---|---|
| WT P. den. | Normal | 8.8 ± 0.2 e-/s (100%) | 14±4/130±10 (58±2%) | 44 ± 2 | 17 ± 4 | |
| WT T. therm. | Normal | 5.5 ± 0.5 e-/min (100%) | - | - | - | |
| T66c (84% T, 15%S, rest:A) In conserved loop with Ca2+ ligands G71C and Y73C | T67cV P. den. | Normal | 6.4 ± 0.2 e-/s (73%) | - | 50±2 | 17 ± 2 |
| T66c (84% T, 15%S, rest:A) In conserved loop with Ca2+ ligands G71C and Y73C | T130cV T.therm. | Normal | 0.77 ± 0.16 e-/min (14%) | - | - | - |
| E77c (76%E,24%D) In conserved loop with Ca2+ ligands G71C and Y73C, forms many hydrogen bonds. | E78cD P. den. | Normal | 5.1 ± 0.1 e-/s (57%) | 3.9±0.1/133±2 (~46%) | 43±5 e) | 19 ± 3 |
| E77c (76%E,24%D) In conserved loop with Ca2+ ligands G71C and Y73C, forms many hydrogen bonds. | E78CF P. den. | This variant did not express | ||||
| E77c (76%E,24%D) In conserved loop with Ca2+ ligands G71C and Y73C, forms many hydrogen bonds. | D141cN T.therm. | This variant was not assembled | ||||
| E77c (76%E,24%D) In conserved loop with Ca2+ ligands G71C and Y73C, forms many hydrogen bonds. | D141cL T.therm. | Normal | 0.99 ± 0.18 e-/min (18%) | - | - | - |
| Q411 98% Q (rest: E,D) | Q394M P. den. | Normal | 5.9 ± 0.2 (67%) | 16±1/145±2 (~57%) | 38±1 | 18 ± 1 |
| Q415 (71% Q, 21%E, 6%W, rest: S,V,Y) | Q398L P. den. | Normal | 8.6 ± 0.4 (98%) | 10±1/144±3 (56±3%) | 31±9 | 21 ± 1 |
a) At pH 7.5 and T = 303K for P. denitrificans, at pH 6.0 and T = 315K for T. thermophilus. The average and the range of at least two different measurements is indicated.
b) At 1 mM CO or O2, pH 7.5 and T = 295K. The average is given with the standard deviation (wildtype, N>6) or the range between two different biological samples (Q398L). For T67cV, E78cD and Q394M the error of the fit is indicated based on ≥3 traces on the same sample.
c) % τ2 indicates the % of the total amplitude that is associated with the second phase at 430 nm.
d) Proton-coupled electron transfer during O2 reduction. The average is given with the standard deviation (wildtype, N = 12) or the range between two different biological samples (Q398L). For T67cV, E78cD and Q394M the error of the fit is indicated based on ≥3 traces at 420 nm, 430 nm and 550 nm of the same sample.
e) This number is the average of those obtained at pH 7, pH 6.75, and pH 8.5 because of a lack of reliable data at pH 7.5 (O2 binding was shown to be pH independent [8]).