toxicity is the so-called metal-catalyzed Haher-Weiss reaction. M"+ + 0; ~ b - l l ++ 0
+
-
-
+
M("-'I+ H,O, M"' OH0,+H,O2-0,+OH-tHO'
+ HO'
In the first step of this scheme, superoxide reacts with metal ions. such as Cu2+or Fe3+.. nresent in the cell in trace . amounts, to form the corresponding reduced metal ions. The second step of the reaction is reduction of hvdroeen neroxide by the redhed metal ion togive hydroxide;on and hydroxsl rndicd. The lntter sueciei is electron-deficient and consequently a powerful, indiscriminant oxidant as well as an initiator of free radical reactions. Certainly the formation of hydroxyl radical in a cell is something to be avoided. However, this mechanism is not an entirely satisfactory explanation of superoxide toxicity since the only role superoxide is playing in this mechanism is as a reducing agent of trace metal ions. The interior of a cell is itself a highly reducing medium. Superoxide does not appear to have any unusual or s ~ e c i a nrooerties l as a reducing aeent when cornoared to dther rebuiing agents, such as &&bate, that are naturally present in the cell. One would therefore exDect that other biological reducing agents in the cell would also be highly toxic because thev would substitute for superoxide in this reaction srheme.'i'he SOlI's would beof no "se in protecting against the formation of hydroxyl rndical by such a route. O f course, there may be other mechanisms whereby superoxide is exerting a toxic effect. For example, it would be difficult to discover if suneroxide were bindine to a soecific site on an essential enzyme and thereby inhigiting it. Cyanide and carbon monoxide would seem to be fairly innocuous species if one were to consider only their chemical reactivities. But their soecific. irreversible bindina to cvtochrome c oxidase and hehoglobin, respectively, make them highly toxic. Another possibility is that superoxide is somehow protonated and that the unrharged H 0 2 dissolves i n mem. species . branes and acts as a promoter of lipid-peroxidation (12). Superoxide could certainly do damage by this mechanism. However, the superoxide dismutase enzymes are cytosolic, i.e., they are dissolved in the aqueous portion of the cell, and therefore the onlv wav thev could Drotect aeainst such reactioni would he toscavengesuperoxidr before it ran get intoa membrane. Since the DK. of HO? is 4.8. verv little of the superoxide will be proionked a t physiologic& pH. Therefore this does not seem like a satisfactory explanation of superoxide toxicity. The mechanism of superoxide toxicity remains a puzzle a t this time. I t is of utmost importance that this problem be solved, however. Dioxygen a t elevated concentrations is highly toxic and for that reason cannot be used therapeutically for long periods of time. The mechanism for the toxicity of dioxygen is unknown. A logical possibility is that dioxygen a t elevated levels leads to the formation of abnormally high concentrations of superoxide. But what are the chemical reactions that account for superoxide toxicity? If superoxide is not toxic. we should be vieorouslv seekine other explanations for dioxygen toxicity and othkr physiological functions for the SOD enzymes! Structure of Cu,Zn-SOD The Richardsons and their coworkers (14) have determined the X-ray crystal structure of the oxidized (i.e., Cu(I1)) form of Cu,Zn-SOD from bovine erythrocytes, the most recent results being based on a 2 A resolution map. There is good reason to believe that the Cu,Zn-SOD'S from other eukarvotic sources have essentiallv the same structure. since the amino acid sequence is highly conserved, especially in the metal-binding region, and the spectroscopic proper-
ties are very similar (I). The X-ray studies show that the protein consists of two identical subunits held together almost entirely by hydrophobic interactions. Each subunit consists of a flattened cylindrical barrel of B-pleated sheet made up of 8 antiparallel chains from which three external loops of irregular structure extend. A schematic drawing of the structure is given in Figure 1. The metal binding region of the protein binds C U ~ and + ZnZt ions in close proximity to each 'ther. The "zinc-binding loop" of the protein, which contains all the amino acid residues involved in zinc coordination, is pinned back to the 0barrel by a disulfide bridge between Cys-55 and Cys-144. This disulfide bond appears to be reinforced by a second bridge formed by hydrogen bonding between the side chain of Are-141 and the backbone carbonvl of Cvs-55. This latter interaction may be related to the "role o? Arg-141 which amears to be an essential residue in the SOD activitv of the protein, a topic discussed in more detail below. Figure 2 is a schematic representation of the copper- and zinc-binding sites. The C U ~ion + is coordinated to four imidazole nitroeen atoms, coming from His-44.46.61. and 118.In addition, a-water molecule is coordinated to copper, giving that ion a distorted sauare-pyramidal eeometrv. The Zn2+ ion is coordinated to imidaidle nitrogens from H i s d l , 69, ~
~~~~~~
Ax
Figure 1. Schematic backbone drawing of Ihe Cu.Zn-SOD subunit. The 6 strands are shown as arrows and the disulfide bridge as a zigzag (from ref. (14)).
~ S N
G L
