The enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O2-. to molecular oxygen and hydrogen peroxide at the active-site Cu ion in a reaction that is rate-limited by diffusion and enhanced by electrostatic guidance. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1). The new crystal structures of human SOD show that amino-acid site chains that are implicated in electrostatic guidance (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu----Gln) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu----Lys) mutant is slower than the equivalent charge neutralization (Glu----Gln) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.

Copper, zinc superoxide dismutase is a dimeric enzyme, and it has been shown that no cooperativity between the two subunits of the dimer is operative. The substitution of two hydrophobic residues, Phe 50 and Gly 51, with two Glu's at the interface region has disrupted the quaternary structure of the protein, thus producing a soluble monomeric form. However, this monomeric form was found to have an activity lower than that of the native dimeric species (10%). To answer the fundamental question of the role of the quaternary structure in the catalytic process of superoxide dismutase, we have determined the solution structure of the reduced monomeric mutant through NMR spectroscopy. Another fundamental issue with respect to the enzymatic mechanism is the coordination of reduced copper, which is the active center. 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-resonance NMR experiments. The solution structure is represented by a family of 36 structures, with a backbone rmsd of 0.81 +/- 0.13 A over residues 3-150 and of 0.56 +/- 0.08 A 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 the oxidized form of human and bovine isoenzymes. The structure contains the classical eight-stranded Greek key beta-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 disorder is present. Significative changes are observed in the conformation of the electrostatic loop, which forms one side of the active site channel and which is fundamental in determining the optimal electrostatic potential for driving the superoxide anions to the copper site which is the rate-limiting step of the enymatic reaction under nonsaturating conditions. In the present monomer, its conformation is less favorable for the 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.

CutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit ␤-sheet formation. Each subunit consists of a ferredoxin-like (␤1␣1␤2␤3␣2␤4) fold with an additional strand (␤5), a C-terminal helix (␣3), and an unusual extended ␤-hairpin involving strands ␤2 and ␤3. The bacterial CutA1 is able to bind copper(II) in vitro through His 2 Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits.CutA1 is a widespread protein of about 12 kDa found in bacteria, plants, and animals, including humans. The protein was originally identified in a gene locus of Escherichia coli called cutA involved in divalent metal tolerance (1). The cutA locus consists of two operons, one containing a single gene encoding a cytoplasmic protein, CutA1, and the other composed of two genes encoding a 50-kDa (CutA2) and a 24-kDa (CutA3) inner membrane proteins. Molecular genetics studies on the E. coli cutA locus showed that some mutations lead to copper sensitivity due to its increased uptake (1). However, the specific function of CutA1 in E. coli is still unknown. On the other hand recent studies from two independent groups highlighted a possible role of mammalian CutA1 in the anchoring of the enzyme acetylcholinesterase (AChE) 1 in neuronal cell membranes (2, 3). CutA1 does not directly interact with AChE (2), but the CutA1 gene is widely expressed in different regions of the brain with an expression pattern that parallels that of AChE (3). In addition CutA1 copurified with AChE from human caudate nucleus (3). CutA1, thus, might provide an intriguing link between copper tolerance in bacteria and a complex process in the brain of the most evolved organisms. The function of CutA1 in plants is still unknown.Copper is a transition metal essential to all organisms since it is involved in many redox reactions and in several biological processes (4). Although essential for cellular metabolism, copper is highly toxic when it exceeds cellular needs and accumulates in the cell. Proteins which bind copper are involved in several human neurological pathologies, such as the Menkes and Wilson diseases, the Alzheimer pathology, and the Creutzfeld-Jacob syndrome (5). For this reason, all organisms must have homeostatic mechanisms that allow the intake of the necessary amount of copper, thus preventing its accumulation beyond the level of toxicity (6, 7); these mechanisms are carried out by proteins t...

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