Dimer Axis Research Articles

Characterization of crystals of a cytochrome oxidase (nitrite reductase) from Pseudomonas aeruginosa by X-ray diffraction and electron microscopy

Cytochrome oxidase from Pseudomonas aeruginosa has been crystallized from 2 m-ammonium sulfate. The crystals occur principally as thin diamond-shaped plates of space group P2 12 12 with unit cell dimensions of 92 Å × 115 Å × 76 Å. Determination of the density of glutaraldehyde-fixed, water-equilibrated crystals (1.167 g/cm 3), coupled with the unit cell volume (804,000 Å 3), indicates that there is one subunit (~63,000 Mr) per asymmetric unit. X-ray diffraction data which were limited to 12 Å resolution due to small crystal size were obtained for the hk0 and 0 kl zones using precession photography. Amplitude and phase data for the hk0, 0 kl, and h0 l zones were obtained from computer-based Fourier analysis of appropriate micrographs recorded from negatively stained microplates and thin sections of larger crystals using minimal beam electron microscopy. For crystals embedded in the presence of tannic acid it was possible to achieve 20 Å resolution which is comparable to the resolution achieved with negative staining of thin crystalline arrays. In addition, unstained electron diffraction on glutaraldehyde-fixed, glucose-stabilized plates was recorded to a resolution of 9 Å. The three-dimensional packing of the cytochrome oxidase dimer in the unit cell has been deduced from computer reconstructed images of the three principal projections along the crystallographic axes. The cytochrome oxidase dimer is located in the unit cell with the dimer axis coincident with a crystallographic 2-fold axis; thus within the resolution of the present data in projection (9 Å) the two subunits are identical, in agreement with biochemical evidence. The crystals have been prepared with the enzyme in the fully oxidized state and upon reduction a progressive cracking of the crystals is observed, possibly due to a conformational change dependent on the oxidation state of the heme iron.

A crystallographic study of alkaline phosphatase at 7.7 Å resolution

An electron density map of crystalline alkaline phosphatase has been calculated to 7.7 Å resolution. The enzyme is present as a dimer on the 2-fold symmetry axis at 0, x, 1 6 in space group P3 121. An orthorhombic form briefly examined is able to contain a tetrameric molecule with at least 2-fold symmetry. The location and environment of two zinc(II) ions has been determined in the dimer by an EDTA treatment of the crystalline enzyme. A small region of electron density located 10 Å from the zinc shifts by 8 Å when the metal is removed. The shift is reversed when zinc is added to the apoenzyme. Hg(II) binds preferentially at the vacant zinc site, and Hg(II) will also exchange with the zinc on the untreated enzyme. The slight 2 Å displacement of Hg from the zinc position may be related to the known inactivity of the mercury enzyme. The two Zn(II) ions are equivalent by symmetry. They are 32 Å apart and 16 Å from the dimer axis. This places them within the molecular envelope, but large channels leading inward from opposite sides of the dimer would allow a substrate molecule to come within 3 Å of a zinc ion. Also, there is space available for a substrate to lie on the dimer axis between the two ions, though 10 Å of protein density would be between the substrate and each zinc ion. In an inconclusive search for active site regions, the enzyme was exposed to p-iodophenylthiophosphate, 2-hydroxy-3-acetomercuri-5-nitrobenzyl-phosphonate, and arsenate ion.