EC Number   |
|---|
   1.7.2.2 | modified form of the enzyme that contains an additional covalent bond between residues Tyr303 and Gln360 in complex with phosphate to 1.45 A resolution, and with sulfite to 1.8 A resolution, structure of unmodified enzyme in complex with nitrite to 1.83 A resolution. Structure reveal the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site |
| 1.7.2.2 | mutant H264N, to 2.15 A resolution. Homodimeric protein, the constellation of His/His and Lys-coordinated c-hemes is indistinguishable from those of the native enzyme |
| 1.7.2.2 | purified NrfA, hanging drop vapor diffusion method, five conditions that result in several different crystal packing motifs, e.g. 0.001 ml of protein solution containing 10 mg/ml of NrfA mixed with 0.001 ml of mother liquor containing 100 mM HEPES, pH 7.5, 20% PEG 10000 and incubated in hanging drop trays with a 1 ml well solution at 4°C, 5-14 days, other conditions, overview. X-ray diffraction structure determination and analysis at 1.7-2.5 A resolution |
| 1.7.2.2 | purified recombinant wild-type and mutant Q263E, 10 mg/ml protein, under aerobic conditions by the vapor diffusion hanging drop method using 20% v/v PEG 10000 in 100 mM Na-HEPES, pH 7.5, 20% ethylene glycol as cryoprotectant, X-ray diffraction structure determination and analysis at 1.74 A and 2.04 A resolution, respectively, molecular replacement |
| 1.7.2.2 | sitting drop vapour diffusion using ammonium sulfate as precipitant. Crystal growth of the NrfHA complex is strongly dependent on the presence of detergent, presumably because of the need to shield the hydrophobic transmembrane helix of NrfH from the solvent. The crystals grown belong to space group I422 |
| 1.7.2.2 | structure at 2.55 A resolution, presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes |
| 1.7.2.2 | Triton X-114 significant improvement in crystal size and quality |
| 1.7.2.2 | UV/Vis spectropotentiometric results yield highly reproducible values for the heme midpoint potentials, which can be assigned to specific hemes in each protomer. Addition of the strong-field ligand cyanide leads to a 70 mV positive shift of the active site's midpoint potential, the cyanide binds to the initially 5-coordinate high-spin heme and triggers a high-spin to low-spin transition. With cyanide present three of the remaining hemes give rise to distinctive and readily assignable EPR spectral changes upon reduction |
| 1.7.2.2 | vapour diffusion method. Crystals belong to space group P2(1)2(1)2(1) with apparent cell parameters of a = 81.47 A, b = 90.84 and c = 294.87 A and contain four molecules of NrfA per asymmetric unit |
