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Title
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X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands.
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Authors
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A.Messerschmidt,
A.Rossi,
R.Ladenstein,
R.Huber,
M.Bolognesi,
G.Gatti,
A.Marchesini,
R.Petruzzelli,
A.Finazzi-Agró.
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Ref.
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J Mol Biol, 1989,
206,
513-529.
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PubMed id
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Abstract
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Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini
have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the
polypeptide chain and the copper ions and their ligands has been built. Crystal
forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr,
respectively, in the asymmetric unit. The crystallographic analysis proceeded by
multiple isomorphous replacement in M2 followed by solvent flattening and
averaging about the local dyad axis. M1 was solved by Patterson search
techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2
were combined and the process of averaging repeated in cycles. An atomic model
was built into the resulting electron density map and refinement initiated. The
current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent
stereo chemistry was maintained, with root-mean-square deviations of bond
lengths and bond angles from average values of 0.02 A and 3.1 degrees,
respectively. Each subunit of about 550 amino acid residues has a globular shape
with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged
sequentially on the polypeptide chain and tightly associated in space. The
folding of all three domains is of a similar beta-barrel type. It is distantly
related to plastocyanin. Each subunit has four copper atoms bound as mononuclear
and trinuclear species. The mononuclear copper has two histidine, a cysteine,
and a methionine ligand and represents the type-1 copper. It is located in the
third domain. The trinuclear cluster has eight histidine ligands. It may be
subdivided into a pair of copper atoms with six histidine ligands arranged
trigonal prismatic. The pair probably represents the type-3 copper. The
remaining copper has two histidine ligands. Its third site of co-ordination is
formed by the pair of copper atoms. The fourth ligand may be OH- represented by
a small protrusion of electron density. This copper probably is the type-2
copper. The symmetry of the trinuclear cluster is C2 and the ligands are
supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a
type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear
cluster probably represents the oxygen binding and electron storage site.
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