Table 3.
Analytical data for WT-NiSOD and mutants.
| NiSOD sample | ESI-MS MWe (calc’d value) | Quaternary Structurec | Ni/protein | EPR as isolated (% Ni(III)) | Kinetics d | E1/2 (mV vs. NHE) | Tm (°C) | |
|---|---|---|---|---|---|---|---|---|
| kcalc x 108 M−1 s−1 @ pH 7.5 [range of O2− concen. (μM)] | Reductive Titration | Oxidative Titration | ||||||
| WT | 18,171.2a (18,169.6) | Hexamer | 0.88 | g = 2.30, 2.23, 2.01 Azz = 24.9G (51(2)) |
7.07 [1.93–5.97] | 290(4) | 279(6) | 84.8 |
| D3A | 13,160.9b (13,157.0) | Hexamer with decreased stability | 1.11 | g = 2.30, 2.24, 2.01 Azz = 24.9G (48(2)) |
2.09 [1.68–5.99] | 308(3) | 290(7) | 73.9 |
| Y9F | 13,183.4b (13,185.0) | Hexamer | 0.72 | g = 2.30, 2.23, 2.01 Azz = 24.9G (53(2)) |
3.71 [1.54–2.38] | 297(5) | 299(8) | 86.3 |
| Y62F | 18,154.1a (18,153.3) | Hexamer | 0.90 | g = 2.30,2.23, 2.01 Azz = 24.9G (48(2)) |
6.42 [2.09–9.8] | ND | ND | 82.4 |
|
Y9F Y62F |
18,137.5a (18,137.2) | Hexamer | 0.74 | g = 2.30,2.23, 2.01 Azz =24.4G (46(2)) |
2.41 [2.18–4.12] | ND | ND | 74.7 |
a
Fusion peptide,
b
Processed NiSOD,
c
from size-exclusion chromatography and/or ESI-MS under non-denaturing conditions (see experimental),
d
Rates determined under first-order conditions; kcalc = kobs/[Ni], where [Ni] = 2 μM. The value of kcalc was determined by averaging the values over the range of [O2−] where there is no apparent decrease in kobs.
e
ESI-MS measurements were conducted under denaturing conditions resulting in monomer MW of processed NiSOD or fusion peptide.