Metal uptake by manganese superoxide dismutase

Abstract

Manganese superoxide dismutase is an important antioxidant defense metalloenzyme that protects cells from damage by the toxic oxygen metabolite, superoxide free radical, formed as an unavoidable by-product of aerobic metabolism. Many years of research have gone into understanding how the metal cofactor interacts with small molecules in its catalytic role. In contrast, very little is presently known about how the protein acquires its metal cofactor, an important step in the maturation of the protein and one that is absolutely required for its biological function. Recent work is beginning to provide insight into the mechanisms of metal delivery to manganese superoxide dismutase in vivo and in vitro.

Fig. 1

Cellular biochemistry of superoxide. Potential sources of superoxide free radicals in living cells (including respiratory chain components, enzymes of the citric acid cycle, and other reduced cofactors, metal ions and metabolites) are shown at the top. Deleterious reactions of reactive oxygen species derived from superoxide (including destruction of Fe-S clusters and DNA mutagenesis) are illustrated below

Fig. 2

Metal delivery mechanisms in vivo. (A) Chaperone-independent metal binding of apo-metalloprotein (Apo-MP) to form holo-metalloprotein (MP) product through direct interaction with the free metal ion. (B) Chaperone-dependent metal binding, mediated by formation of a metallochaperone (MC) complex, permitting targeted delivery of the metal ion to a specific apo-metalloprotein via a heterdimeric intermediate.

Fig. 3

Pathways for SOD metallation. (A) Eukaryotic (yeast) pathways for metal delivery to Cu,Zn-SOD (Sod1) and mitochondrial MnSOD (Sod2) are illustrated. Cu and Zn enter the cell through specific transporters. Metal delivery to the Sod1 apoprotein has been shown to involve metallochaperones (including the copper chaperone for superoxide dismutase, CCS). Mn uptake involves a specific transporter (Smf1), and intracellular Mn is trafficked to the vacuole (V). A specific pump (Smf2) mobilizes Mn reserves for delivery to the mitochondrial matrix, where it binds to the Sod2 apoprotein. (B) Prokaryotic pathways for metal delivery to NiSOD (SodN), MnSOD (SodA) and FeSOD (SodB). Specific transporters in the cell membrane (NikD, MntH, FeoB) mediate uptake of the metal ions into the cytosol for delivery to SOD apo-proteins via either chaperone-dependent or chaperone-independent mechanisms.

Fig. 4

Timeline for metal binding by metalloproteins. In principle, metal binding may occur at any point during biosynthesis of the protein, including (A) co-translational metal binding to the nascent polypeptide chain emerging from the ribosome; (B) post-translational metal binding to the isolated, fully folded subunit prior to assembly of the multimer; or (C) post-translational metal binding to the completely folded and assembled multimer.

Fig. 5

Differential scanning calorimetry analysis of metal binding affinity for manganese superoxide dismutase. (A) DSC endotherms for global unfolding of Escherichia coli MnSOD complexes, including (1) apo-MnSOD, (2) reduced Mn(II)₂-MnSOD, and (3) oxidized Mn(III)2-MnSOD. (B) Color-coded thermodynamic box diagram corresponding to structural equilibria represented in the DSC endotherms (A).

Fig. 6

Manganese and iron complexes of MnSOD. (Left) The active site of native E. coli Mn₂-MnSOD contains a trigonal bipyramidal 5-coordinate metal center. (Based on PDB ID 1VEW). (Right) The corresponding view of the Fe-substituted enzyme (Fe₂-MnSOD), showing tetragonal 6-coordination of the metal cofactor resulting from ligation by a second solvent-derived hydroxide. The hydroxide adduct blocks the substrate-access gateway to the active site. (Based on PDB ID 1MMM). Prepared with the program molscript [106].

Fig. 7

Fluorimetric assay for metal uptake by apo-manganese superoxide dismutase. (Top, Right) A single subunit of E. coli MnSOD with its buried metal center is shown, with ball-and-stick rendering of the clustered tryptophan side chains. (Based on PDB ID 1VEW) (Top, Left) Jablonski diagram illustrating the predicted fluorescence quenching mechanism for tryptophan residues in the presence of a bound metal ion. EX, excitation; EM, emission; Q, quenching by Förster energy transfer and nonradiative decay. (Bottom) Fluorescence timecourse for Co²⁺ binding by apo-MnSOD in 20 mM MOPS (pH 7.6), 37°C. Resolution of initial amplitude (A₀) and total amplitude (A_TOT) for the biphasic uptake kinetics is shown.

Fig. 8

Temperature and pH dependence of apo-MnSOD metal uptake. (Top) Temperature profile for ratio of initial to total amplitude (A₀/A_TOT) measured from the biphasic kinetics of Co²⁺ uptake by E. coli apo-MnSOD in 20 mM MOPS pH 7. (Bottom) pH dependence for ratio of initial to total amplitude (A₀/A_TOT) for uptake of Co²⁺ by E. coli apo-MnSOD at 37°C.

Fig. 9

Hypothetical models for gated metal binding mechanism. (A) Conformational gating associated with a temperature-dependent conformational equilibrium between closed and open states. Metal binding occurs exclusively in the open state to irreversibly form the holoprotein product. (B) Structurally gating associated with temperature-dependent dimer dissociation, with metal binding to the isolated monomeric subunits followed by irreversible assembly of the dimeric holoprotein product.

Fig. 10

Covalently cross-linked MnSOD variants. Disulfide engineering methods have been used to introduce cysteine pairs into MnSOD, which were covalently cross-linked to form disulfides by oxidation or bridged by treatment with a bifunctional thiol reagent (dBmBr). The locations of the cross-links are mapped onto the native MnSOD structure (PDB ID 1VEW). Prepared with the programs molscript [106] and raster3d [107].

Fig. 11

Hypothetical structures for apo-MnSOD conformational gating states. (A) Closed gate conformation for apo-MnSOD, based on the structure of the metallated holoenzyme (PDB 1vew). The dashed line represents the subunit interface. His171 serves as a metal ligand in the holoenzyme. Rotation of this side chain with reorientation of the interface-spanning Glu170 from the opposing subunit would open a large access channel into the metal binding region in the interior of the protein (B). Two additional residues, Tyr173 and Arg180, may serve to stabilize the protein in the open conformation. Prepared with the programs molscript [106] and raster3d [107].