Solution Structure of Reduced Monomeric Q133M2 Copper, Zinc Superoxide Dismutase (SOD). Why Is SOD a Dimeric Enzyme?
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- 作者：Lucia Banci ; Marco Benedetto ; Ivano Bertini ; Rebecca Del Conte ; Mario Piccioli ; and Maria Silvia Viezzoli
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：August 25, 1998
- 年：1998
- 卷：37
- 期：34
- 页码：11780 - 11791
- 全文大小：240K
- 年卷期：v.37,no.34(August 25, 1998)
- ISSN：1520-4995
文摘
Copper, zinc superoxide dismutase is a dimeric enzyme, and it has been shown that nocooperativity between the two subunits of the dimer is operative. The substitution of two hydrophobicresidues, Phe 50 and Gly 51, with two Glu's at the interface region has disrupted the quaternary structureof the protein, thus producing a soluble monomeric form. However, this monomeric form was found tohave an activity lower than that of the native dimeric species (10%). To answer the fundamental questionof the role of the quaternary structure in the catalytic process of superoxide dismutase, we have determinedthe solution structure of the reduced monomeric mutant through NMR spectroscopy. Another fundamentalissue with respect to the enzymatic mechanism is the coordination of reduced copper, which is the activecenter. The three-dimensional solution structure of this 153-residue monomeric form of SOD (16 kDa)has been determined using distance and dihedral angle constraints obtained from 13C, 15N triple-resonanceNMR experiments. The solution structure is represented by a family of 36 structures, with a backbonermsd of 0.81 ± 0.13 Å over residues 3-150 and of 0.56 ± 0.08 Å over residues 3-49 and 70-150.This structure has been compared with the available X-ray structures of reduced SODs as well as with theoxidized form of human and bovine isoenzymes. The structure contains the classical eight-stranded Greekkey 
-barrel. In general, the backbone and the metal sites are not affected much by the monomerization,except in the region involved in the subunit-subunit interface in the dimeric protein, where a large disorderis present. Significative changes are observed in the conformation of the electrostatic loop, which formsone side of the active site channel and which is fundamental in determining the optimal electrostaticpotential for driving the superoxide anions to the copper site which is the rate-limiting step of the enymaticreaction under nonsaturating conditions. In the present monomer, its conformation is less favorable forthe diffusion of the substrate to the reaction site. The structure of the copper center is well-defined;copper(I) is coordinated to three histidines, at variance with copper(II) which is bound to four histidines.The hydrogen atom which binds the histidine nitrogen detached from copper(I) is structurally identified.
-barrel. In general, the backbone and the metal sites are not affected much by the monomerization,except in the region involved in the subunit-subunit interface in the dimeric protein, where a large disorderis present. Significative changes are observed in the conformation of the electrostatic loop, which formsone side of the active site channel and which is fundamental in determining the optimal electrostaticpotential for driving the superoxide anions to the copper site which is the rate-limiting step of the enymaticreaction under nonsaturating conditions. In the present monomer, its conformation is less favorable forthe diffusion of the substrate to the reaction site. The structure of the copper center is well-defined;copper(I) is coordinated to three histidines, at variance with copper(II) which is bound to four histidines.The hydrogen atom which binds the histidine nitrogen detached from copper(I) is structurally identified.