Metal-substituted zinc proteins

Varying ratios of the ligands 7V-(2-thiophenyl)-2,5-dimethylpyrrole and V-methylimidazole were used to form tetrahedral zinc complexes with S4, S3N, and S2N2 coordination spheres. X-ray structural analyses and IR spectra were recorded for all compounds and the relevance to zinc finger proteins was discussed. The comparison to cobalt and cadmium structures showed only minor differences, supporting the theory that changes on substituting these metals into zinc proteins would be minor.538... 

Copper(II)-substituted zinc proteins are generally inactive with respect to the natural and most artificial substrates (Table 2.4). In model compounds cop-per(II) is often principally four-coordinate, with at most two more ligands present at metal-ligand distances that are longer than normal coordination bonds. As a consequence, tbe ability of zinc to switch between four- and five-coordinate species without any appreciable barrier and with usual metal-donor distances is not mimicked by copper. Furthermore, binding at the four principal coordination positions is generally stronger for copper than for zinc. It follows that substrates may have slow detachment kinetics. These properties are unfavorable for catalysis. 

A commonexperimental strategy for studying electron transfers between proteins uses a metal-substituted heme protein as one of the reactants. In particular, the substitution of zinc for iron in one of the porphyrin redox centers allows facile initiation of electron transfer through photoexcitation of the zinc porphyrin (ZnP). The excited zinc porphyrin, ZnP in Equation (6.32),... 

In homo-dinuclear systems, such as two copper(ll) ions, no large effects are expected on the electron relaxation rates as the two metal ions relax at the same rate. However, some other relaxation mechanisms are operative, giving rise to faster electron relaxation rates (dementi and Luchinat, 1998). Consequently, nuclear relaxation is slower than in single copper(ll) systems. Several examples from model complexes are available (Brink et al., 1996 Murthy et al., 1997), as well as from a copper(ll)-substituted zinc enzyme, the aminopeptidase from Aeromonas proteolytica (Holz et al., 1998). In contrast, few NMR studies on native copper proteins containing two coupled copper (II) ions have been reported so far (Bubacco cf a/., 1999). 

In vitro, both metals in the active site of SOD can be reversibly removed. This behavior has been elegantly expanded to produce a variety of metal-substituted derivatives. Co, Ni, and Ag derivatives, where the added metal ion may occupy the copper site or the zinc site (and in some cases both), have proven especially valuable in providing new spectroscopic probes of metal-site structure and reactivity in Cu, Zn SOD. Application of in vitro metal substitution to several of the FALS wild-type-like mutants has shown that these variants readily misfold and often exhibit altered metal ion binding. In addition to the in vitro metal studies, recent work has centered on the mechanism of in vivo copper incorporation. CCS is a copper chaperone protein that forms a heterodimer with Zn-loaded SOD to insert copper and activate the enzyme (see Metallochaperones Metal Ion Homeostasis). 

Skin sensitization is believed to occur as a result of nickel binding to proteins (particularly on the cell surface) and hapten formation. Essentially, the body perceives the nickel-protein complex as foreign and mounts an immune reaction to it. For example, sweat may react with the nickel in plated jewelry that comes in direct contact with skin dissolved metal may penetrate and react with proteins in the skin and lead to immune sensitization. Nickel may substitute for certain other metals (especially zinc) in metal-dependent enzymes, leading to altered protein function. High nickel content in serum and tissue may interfere with both copper and zinc metabolism. It also readily crosses the cell membrane via calcium channels and competes with calcium for specific receptors. 

All the above structural and kinetic information obtained under a variety of conditions with different metal ions can be used to propose a catalytic cycle for carbonic anhydrase (Figure 2.21). As shown by studies on the pH-dependent properties of native and metal-substituted CAs, both type-I and type-II proteins have two acidic groups, the zinc-coordinated water and a free histidine. At... 

Metallothioneins (MT) are unique 7-kDa proteins containing 20 cysteine molecules bounded to seven zinc atoms, which form two clusters with bridging or terminal cysteine thiolates. A main function of MT is to serve as a source for the distribution of zinc in cells, and this function is connected with the MT redox activity, which is responsible for the regulation of binding and release of zinc. It has been shown that the release of zinc is stimulated by MT oxidation in the reaction with glutathione disulfide or other biological disulfides [334]. MT redox properties led to a suggestion that MT may possesses antioxidant activity. The mechanism of MT antioxidant activity is of a special interest in connection with the possible antioxidant effects of zinc. (Zinc can be substituted in MT by some other metals such as copper or cadmium, but Ca MT and Cu MT exhibit manly prooxidant activity.)... 

Table I lists isomorphous replacements for various metalloproteins. Consider zinc enzymes, most of which contain the metal ion firmly bound. The diamagnetic, colorless zinc atom contributes very little to those physical properties that can be used to study the enzymes. Thus it has become conventional to replace this metal by a different metal that has the required physical properties (see below) and as far as is possible maintains the same activity. Although this aim may be achieved to a high degree of approximation [e.g., replacement of zinc by cobalt(II)], no such replacement is ever exact. This stresses the extreme degree of biological specificity. The action of an enzyme depends precisely on the exact metal it uses, stressing again the peculiarity of biological action associated with the idiosyncratic nature of active sites. (The entatic state of the metal ion is an essential part of this peculiarity.) Despite this specificity, the replacement method has provided a wealth of information about proteins that could not have been obtained by other methods. Clearly, there will often be a compromise in the choice of replacement. Even isomorphous replacement that should retain structure will not necessarily retain activity at all. However, it has become clear that substitutions can be made for structural studies where the substituted protein is inactive (e.g., in the copper proteins and the iron-sulfur proteins). It is also possible to substitute into metal coenzymes. Many studies have been reported of the...

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