Metal Ions into Metalloproteins

As we will also see in Chapter 8, free copper levels are extremely low within cells because the copper is bound to a family of metallochaperones which are subsequently involved in the incorporation of copper into copper-containing proteins. The mechanism proposed for copper insertion into the Cu/Zn superoxide dismutase, SOD 1, is presented in Fig. 4.7, and appears to use an already-preformed Cu-binding site. The copper chaperone CCS acquires copper as Cu from a copper transporter and then docks with the reduced dithiol form of SODl (steps I 

Chaperones were persons who, for the sake of propriety, accompanied young unmarried ladies in public, as guide and protector. 

We discuss briefly now how metals are incorporated into porphyrins and corrins to form haem and other metallated tetrapyrroles, and how Fe—S clusters are synthesised. 

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Injury to cells and tissues may enhance the toxicity of the active oxygen species by releasing intracellular transition metal ions (such as iron) into the surrounding tissue from storage sites, decompartmentalized haem proteins, or metalloproteins by interaction with delocalized proteases or oxidants. Such delocalized iron and haem proteins have the capacity to decompose peroxide to peroxyl and alkoxyl radicals, exacerbating the initial lesion. 

Metallothionein was first discovered in 1957 as a cadmium-binding cysteine-rich protein (481). Since then the metallothionein proteins (MTs) have become a superfamily characterized as low molecular weight (6-7 kDa) and cysteine rich (20 residues) polypeptides. Mammalian MTs can be divided into three subgroups, MT-I, MT-II, and MT-III (482, 483, 491). The biological functions of MTs include the sequestration and dispersal of metal ions, primarily in zinc and copper homeostasis, and regulation of the biosynthesis and activity of zinc metalloproteins. 

While recognizing that this is a supreme example of reductionist simplification, it nonetheless allows us to situate the three model systems that we will consider here, because the mechanisms of metal assimilation are often significantly different. Finally, once the metal ions have been assimilated, they must be incorporated into the corresponding proteins, we have already presented in Chapter 3 a brief consideration of some of the ways in which metal centres, whether individual metal ions or more complex metal clusters, are engineered into their metalloprotein targets. 

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...

Metal labels have been proposed to resolve problems connected with enzymes. Metal ions [13-16], metal-containing organic compounds [17,18], metal complexes [19-21], metalloproteins or colloidal metal particles [22-28] have served as labels. Spectrophotometric [22,25], acoustic [25], surface plasmon resonance, infrared [24] and Raman spectroscopic [28] methods, etc. were used. A few papers have been dealing with electrochemical detection. However, electrochemical methods of metal label detection may be viewed as very promising taking into account their high sensitivity, low detection limit, selectivity, simplicity, low cost and the availability of portable instruments. 

The artificial intelligence-superexchange method in which the details of the electronic structure of the protein medium are taken into account was used for estimating the electronic coupling in the metalloproteins (Siddarth and Marcus, 1993a,b,c). Fig.2.11 demonstrates a correlation of experimental and calculated ET rate constants for cytochrome c derivatives, modified by Ru complexes. The influence of the special mutual orientation of the donor and acceptor orbitals in Ru(bpy)2im HisX-cytochrome c on the rate of electron transfer was analyzed by the transition amplitude methods (Stuchebrukhov and Marcus, 1995). In this reaction the transferring electron in the initial and the final states occupies the 3d shell of the Fe atom and the 4d shell of Ru, respectively. It was shown that the electron is localized on t2g subshells of the metal ions. Due to the near-... 

COXl 7 copper carrying molecule for the mitochondria IMS intermembrane space of mitochondria, lying between the inner and outer membranes of this organelle Metal chaperone molecule that binds a specific metal and helps insert this ion into the metal binding site of a metalloprotein... 

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

The zinc and copper Lons that are absorbed from the diet enter the portal vein, where they are weakly bound to plasma albumin. This situation Is reminiscent of that with dietary medium-chain fatty adds. The metal ions enter various tissues of the body, where they are assimilated into various metalloproteins. 

In Chapter 2, we explained the basic notions involved in the coordination chemistry of metal ions. We now consider potential ligands, which could be involved in binding metals in metalloproteins. We already defined ligand binding as the affinity of the metal ion for any atom, group, or molecule that is attached to the central metal ion. We can divide them into three categories ... 

If it is assumed that the metal ion concerned is present in all four of the states mentioned in Chapter 3, then it will be present as inert metalloprotein or a solid material, and then three equilibrium states of circulating labile protein in equilibrium with low molar mass complexes in equilibrium with exceedingly low concentrations of aquated metal ion. Such a concept can be modelled in respect of its labile equilibrium and the ability of sequestering drugs to remove the metal ion from the labile protein into low molar mass form. Thereafter its destiny is determined by whether the material has charges for its low molecular mass (l.m.m.) species or whether these are uncharged, and therefore potentially bioavailable through a cell membrane. 

Binding those metal ions in a metalloprotein usually prevents them from entering into these types of reactions. For example, transferrin, the iron-transport enzyme in serum, is normally only 30 percent saturated with iron. Under conditions of increasing iron overload, the empty iron-binding sites on transferrin are observed to fill, and symptoms of iron poisoning are not observed in vivo until after transferrin has been totally saturated with iron. Ceruloplasmin and metallothionein may play a similar role in preventing copper toxicity. It is very likely that both iron and copper toxicity are largely due to catalysis of oxidation reactions by those metal ions. 

Hausinger, R. P. Mechanisms of metal ion incorporation into metalloproteins. BioFactors 2 (1990), 179-184. 

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