X-Ray Crystallographic Studies on Electron Transfer Proteins; Rubredoxin from Pyrococcus furiosus, Nitrogenase MoFe from Azotobacter vinelandii and Ru(2,2'-bppy)₂(imd)His83 Azurin from Pseudomonas aeruginosa

Citation

Day, Michael W. (1996) X-Ray Crystallographic Studies on Electron Transfer Proteins; Rubredoxin from Pyrococcus furiosus, Nitrogenase MoFe from Azotobacter vinelandii and Ru(2,2'-bppy)₂(imd)His83 Azurin from Pseudomonas aeruginosa. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Y70P-TJ52. https://resolver.caltech.edu/CaltechTHESIS:11062019-175229018

Abstract

The x-ray crystal structure of the oxidized and the reduced forms rubredoxin from Pyrococcus furiosus, a hyperthermophilic marine Archae, have been solved by molecular replacement and refined by the method of restarined least squares to a maximum resolution of 1.1 Å for the oxidized form and 1.5 Å for the reduced form. The oxidized form of the protein crystallizes in the orthorhombic space group P 2₁2₁2₁ with unit cell dimensions of a = 33.8 Å, b = 34.6 Å, c = 43.4 Å and V = 50,755 ų. The reduced form crystallizes in the same space group with the nearly identical unit cell dimensions of a = 33.8 Å, b = 34.5 Å, c = 43.2 Å and V = 50,375 ų. Data on both forms was collected at -161°C. Three refinement packages were used in the refinement and the results from each arc discussed as are the possible determinants of the thermal stability. Refinement of the oxidized form (414 protein atoms and 104 solvent oxygens) with TNT or XPLOR resulted in a crystallographic residual of approximately 17% and a model with rms deviations of bond distances and angles from target values of approximately 0.015 Å and 2.5° respectively. Refinement of the oxidized form with SHELXL-93 resulted in a model with 132 solvent oxygens and an R = 13.9% (R_free = 17.2%) and GOF = 1.08. The rms deviation from the target values for bond distance and angles are 0.014 Å and 1.75° respectively. Refinement of the reduced form with TNT (110 solvent oxygens) or SHELXL-93 (including 130 solvent oxygen atoms) results in an R-factor of approximately 17% and the geometry of the model deviates from the target values by approximate rms values of 0.022 Å for the bond distances and 3.0° for the bond angles.

The x-ray crystal structure of the MoFe nitrogenasc protein from Azotobcacter vinelandii has been refined against data collected at the Stanford Synchrotron Radiation Laboratory (SSRL). The data extends to a maximum resolution of 2.2 Å and two packages were used in the restrained least squares refinement. Refinement of the model (including 625 solvent oxygens) with TNT or XPLOR yielded a crystallographic residual of less than 18% and a model with bond distances and angles deviate from the target values by rms values of 0.02 Å and 2.5° respectively.

The x-ray crystal structure of the Ru(2,2'-bppy)₂(imd)His83 azurin from Pseudomonas aeruginosa has been solved and refined by the method of restrained least squares to a limiting resolution of 2.5 Å. The labeled protein crystalizes in the monoclinic space group C 2 with a = 100.6 Å, b = 35.4 Å, c = 74.7 Å, b = 106.5° V = 255,069 ų and Z = 8. Data was collected at -161°C to a maximum resolution of 2.3 Å yielding a data set that is 82% complete containing 11,083 reflections. Refinement in TNT (including 150 solvent oxygen atoms) resulted in an R-factor of 17.3% with rms deviations in the model bond distances and angles from ideal values of 0.026 Å and 3.09° respectively.

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