Table 3. Properties of cNOR variants in the heme propionate and Ca2+ binding region.
New data is shown in bold and compared to earlier published results from Refs. [3,7,9,11,13]. Conservation and location is taken from or calculated as described in Ref. [7].
| Res. In Ps. aer. (conservation) Location | Mutation in P. den. / T. therm. | Spectra | Maximum turnover activity NO (% of WT activity) a) | CO b) binding τ1 (μs)/ τ2 (μs) (% τ2) c) 0 | O2 b) binding (μs) 0 | ETPT d) τ (ms) at pH 7.5 |
|---|---|---|---|---|---|---|
| WT P. den | normal | 100% | 14±4/130±10 (58±2%) | 44 ± 2 | 17 ± 4 | |
| E70 C (80% E, rest N,H,Q,S,F) In conserved loop with Ca2+ ligands | E71CQ P. den | α-region slightly altered e) | as background 0% | 13±1/157±1 (67±1%) | 85 ± 4 | - |
| E70 C (80% E, rest N,H,Q,S,F) In conserved loop with Ca2+ ligands | E71CD P. den | α-region altered e) less pure | 27±3% not sigmoidal | 40±20/160±20 (50±9%) | 48 ± 4 | (8 ± 1)f) |
| Y73C (99% Y, rest R) Ca2+ ligand | Y74CS P. den | α-region slightly altered e) | 53±3% | 14±10/150±30 (55±1%) | 43 ±3 | 13 ± 2 |
| Y73C (99% Y, rest R) Ca2+ ligand | Y74CF P. den | normal | 53±3% | 16±1/121±2 (55±1%) | 48 ±7 | 29 ± 1 |
| R134 (96%, rest NIML) Next to Ca2+ ligand E135 | R121Q P. den | normal | 9.4±0.3% | 30±10/200±20 (60±10%) | 50 ± 7 | (32± 7)f) |
| E135 (88%E,9%K, rest: H,S,V,P) Ca2+ ligand | E122A P. den. | normal | 14% 35±6% | 5–10/50–100 (~50%) 19±4/140±10 (57±3%) | 40, 55±5 | 17 ± 1 |
| E135 (88%E,9%K, rest: H,S,V,P) Ca2+ ligand | E122D P. den. | normal | 83% 30±8% | 5–10/50–100 (~50%) 30±10/170±30 (52±6%) | ~40, 80±10 | ~10 (30±10)f) |
| E135 (88%E,9%K, rest: H,S,V,P) Ca2+ ligand | E122Q P. den. | normal | 1% | 5–10/50–100 (~50%) | ~40 | - |
| E138 (96%rest: D) In loop with Ca2+ ligand | E125D P. den | altered less pure | 2%, <10% | 30±8/180±20 (62±4%) | 47±2 | - |
| E138 (96%rest: D) In loop with Ca2+ ligand | E125Q P. den | - | 13% | - | ~40 | - |
| E138 (96%rest: D) In loop with Ca2+ ligand | E125A P. den | normal | 4–7% | - | ~40 | - |
| D198 (96%D 2%E, rest: A,S,Q) In proton transfer PW1, H-bonds with R134, K53C | D185E P. den | normal | 12±1% | 29±1/146±6 (54±2%) | 52 ± 4 | (33±2)g) |
| D198 (96%D 2%E, rest: A,S,Q) In proton transfer PW1, H-bonds with R134, K53C | D185N P. den | normal | <<10% | 21±1/147±1 (~58%) | 62 ± 1 | (18±6)g) |
| D198 (96%D 2%E, rest: A,S,Q) In proton transfer PW1, H-bonds with R134, K53C | D185A P. den | normal | as background 0% | 34±1/145±2 (~51%) | 64 ± 3 | - |
| N335 (100% N) H-bond D-prop. heme b3 | N322L P. den | altered e) | 51±6% | 24±6/180±10 (70±10%) | 58 ± 4 | (21±4)f) |
| N335 (100% N) H-bond D-prop. heme b3 | N335L T. therm. | normal h) | 15% | N.D. | N.D. | - |
| H339 (99%H, 1xR) H-bond D-prop. heme b3 | H326F P. den | altered e) | as background 0% | 58±1/178±4 (39±2%) | 40 ± 3 (+slower fraction) | - |
| H339 (99%H, 1xR) H-bond D-prop. heme b3 | H339F T. therm. | normal h) | 0% | N.D. | N.D. | - |
a) At pH 7.5 and T = 295K for P. denitrificans, at pH 6.0 and T = 315K for T. thermophilus. For new data 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, E122A, N322L and E71cD: N = 3) or the range between two different biological samples, except for D185N and E71cQ where 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. The average is given with the standard deviation (wildtype: N = 12, N322L and E71cD: N = 3) or the range between two different biological samples except for D185N where the error of the fit is indicated based on ≥3 traces at 430 nm and 550 nm of the same sample.
e) Especially after incubation with CO, see S2 Fig.
f) The fit of these variants is based only on the 430 nm and 550 nm data, since they did not have any signal for the ETPT at 420 nm.
g) Not fitted in Ref. [7] because of absence signal for ETPT at 420 nm and small signals at 430 nm and 550 nm. Now fitted with 550 nm and 430 nm data as described for the other variants in this table.
h) Oxidized and reduced spectra only [13].