Tài liệu Báo cáo khoa học: The solution structure of reduced dimeric copper zinc superoxide

The solution structure of reduced dimeric copper zinc superoxide

dismutase

The structural effects of dimerization

Lucia Banci, Ivano Bertini, Fiorenza Cramaro, Rebecca Del Conte and Maria Silvia Viezzoli

Department of Chemistry and Centro Risonanze Magnetiche, University of Florence, Italy

The solution structure of homodimeric Cu2Zn2 superoxide

dismutase (SOD) of 306 aminoacids was determined on a

13C, 15N and 70% 2

H labeled sample. Two-thousand eight￾hundred and five meaningful NOEs were used, of which 96

intersubunit, and 115 dihedral angles provided a family of 30

conformers with an rmsd from the average of 0.78 ± 0.11

and 1.15 ± 0.09 A˚ for the backbone and heavy atoms,

respectively. When the rmsd is calculated for each subunit,

the values drop to 0.65 ± 0.09 and 1.08 ± 0.11 A˚ for the

backbone and heavy atoms, respectively.

The two subunits are identical on the NMR time scale, at

variance with the X-ray structures that show structural dif￾ferences between the two subunits as well as between dif￾ferent molecules in the unit cell. The elements of secondary

structure, i.e. eight b sheets, are the same as in the X-ray

structures and are well defined. The odd loops (I, III and V)

are well resolved as well as loop II located at the subunit

interface. On the contrary, loops IV and VI show some

disorder. The residues of the active cavity are well defined

whereas within the various subunits of the X-ray structure

some are disordered or display different orientation in dif￾ferent X-ray structure determinations. The copper(I) ion and

its ligands are well defined. This structure thus represents a

well defined model in solution relevant for structure–func￾tion analysis of the protein. The comparison between the

solution structure of monomeric mutants and the present

structure shows that the subunit–subunit interactions in￾crease the order in loop II. This has the consequences of

inducing the structural and dynamic properties that are

optimal for the enzymatic function of the wild-type enzyme.

The regions 37–43 and 89–95, constituting loops III and V

and the initial part of the b barrel and showing several

mutations in familial amyotrophis lateral sclerosis (FALS)-

related proteins have a quite extensive network of H-bonds

that may account for their low mobility. Finally, the con￾formation of the key Arg143 residue is compared to that in

the other dimeric and monomeric structures as well as in the

recently reported structure of the CCS–superoxide dismu￾tase (SOD) complex.

Keywords: superoxide dismutase; solution structure; dimeric

protein; NMR; FALS.

Cu2Zn2SOD is a well known homodimeric enzyme of

32 000 Da that catalyzes the dismutation of the superoxide

radical to hydrogen peroxide and oxygen through a two step

reaction [1–5]:

Cu2þ þ Oÿ

2 ! Cuþ þ O2

Cuþ þ Oÿ

2 ! Cu2þ O2ÿ

2

ÿ
ƒƒƒƒ!2Hþ

Cu2þ þ H2O2

The active site of each subunit contains both a zinc and a

copper ion, the latter being the site of the reaction. Copper

occurs in the oxidized and in the reduced state, both of

which are necessary for the function. The X-ray structure of

the oxidized form has been available since 1982 for the

bovine enzyme [6,7] and several other structures have

become available [8–18]. Reduced state structures are also

available although the picture is less clear-cut around

the copper-binding site [19–21]. Certainties on the protona￾tion of His63, which bridges Cu and Zn in the oxidized form

but is protonated in the reduced form, come from 1

H NMR

studies [22–25]. Eventually, monomeric forms were

obtained through site-specific mutagenesis and the NMR

solution structure [26,27] as well as the crystal structure [28]

of the reduced form were reported. Also the backbone

mobility of the monomeric state was investigated and

compared with that of the dimeric species and it was

concluded that, as far as motions in the ps to ns timescale

are concerned, the region consisting of residues 131–142,

which forms one side of the active site channel, is less mobile

in the monomeric mutant than in the dimeric wild-type

protein; structural fluctuations in this region have been

suggested to play a role in assisting the superoxide anion in

sliding towards the active site [29,30]. Moreover, the regions

consisting of residues 47–59, 76–86 and 151–153, which are

Correspondence to I. Bertini, Department of Chemistry and Centro

Risonanze Magnetiche, University of Florence, Via Luigi Sacconi 6,

50019 Sesto Fiorentino, Italy.

Fax: + 39 055 4574271, Tel.: + 39 055 4574272,

E-mail: [email protected]

Abbreviations: SOD, superoxide dismutase; Q133M2SOD, F50E/

G51E/E133Q monomeric mutant superoxide dismutase; FALS,

familial amyotrophis lateral sclerosis; M4SOD, F50E/G51E/V148K/

I151K monomeric mutant superoxide dismutase; CCS, yeast copper

chaperone for superoxide dismutase; TPPI, time proportional phase

increments.

Note: The PDB ID code for the solution structure of homodimeric

Cu2Zn2 superoxide dismutase is 1L3N.

(Received 5 October 2001, revised 4 February 2002, accepted 16

February 2002)

Eur. J. Biochem. 269, 1905–1915 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02840.x