Proteins metal cofactors

Copper is an essential trace element. It is required in the diet because it is the metal cofactor for a variety of enzymes (see Table 50—5). Copper accepts and donates electrons and is involved in reactions involving dismu-tation, hydroxylation, and oxygenation. However, excess copper can cause problems because it can oxidize proteins and hpids, bind to nucleic acids, and enhance the production of free radicals. It is thus important to have mechanisms that will maintain the amount of copper in the body within normal hmits. The body of the normal adult contains about 100 mg of copper, located mostly in bone, liver, kidney, and muscle. The daily intake of copper is about 2—A mg, with about 50% being absorbed in the stomach and upper small intestine and the remainder excreted in the feces. Copper is carried to the liver bound to albumin, taken up by liver cells, and part of it is excreted in the bile. Copper also leaves the liver attached to ceruloplasmin, which is synthesized in that organ. 

Heavy metals with no known biological function, such as aluminum, arsenic, lead, and mercury, are nonessential metals.4-5 These metals are toxic because they can irreversibly bind to enzymes that require metal cofactors. Toxic metals readily bind to sulfhydryl groups of proteins.6-7 In fact,... 

Probably the most effective use of XRF and TXRF continues to be in the analysis of samples of biological origin. For instance, TXRF has been used without a significant amount of sample preparation to determine the metal cofactors in enzyme complexes [86]. The protein content in a number of enzymes has been deduced through a TXRF of the sulfur content of the component methionine and cysteine [87]. It was found that for enzymes with low molecular weights and minor amounts of buffer components that a reliable determination of sulfur was possible. In other works, TXRF was used to determine trace elements in serum and homogenized brain samples [88], selenium and other trace elements in serum and urine [89], lead in whole human blood [90], and the Zn/Cu ratio in serum as a means to aid cancer diagnosis [91]. 

Three other proteins with similar domain structure as that of FprA were reported in other bacteria (WasserfaUen et al. 1995 Gomes et al. 1997, 2000). The recombinant CthFprA and CthHrb, overexpressed in E. coli, were purified and characterized. Both FprA and Hrb were found to be present as dimers. Metal/cofactor analysis of the purified proteins revealed the presence of 2 mol each of iron and flavin (FMN) per mole dimer of Hrb and 4 mol of iron and 2 mol FMN per mole dimer of FprA. The EPR spectra of the purified proteins indicated that iron is present in a di-iron center in FprA and as a Fe(Cys)4 cluster in Hrb. 

Mammalian Cell Protease Inhibitor CocktaiL These should contain AEBSF, pepstatin A, E-64, bestatin, leupeptin, and aprotinin. (Metal chelators can be added to suppress the activity of calcium ion-dependent proteases such as calpain. Again, one must determine whether the protein or enzyme being purified does not require a divalent metal cofactor for stabihty or activity.)... 

The multiprotein complex methane monooxygenase (MMO) serves meth-anotrophs to convert methane to methanol. It can be either soluble (sMMO) or membrane bound ( particulate , pMMO) and it typically consists of three components, a reductase (MMOR), a component termed protein B (MMOB) and a hydroxylase denoted MMOH. The nature of the metal cofactors in the latter component are reasonably well understood for sMMO as will be discussed in the non-heme iron section. For the pMMO of Methylococcus capsulatus an obligate requirement for copper was shown. As reported in reference 1 a trinuclear Cu(II) cluster was discussed128 but the number and coordination of coppers still is a matter of continuing investigation since then. 

A previously created receptor description file (RDF) can be used (Fig. 1), or a new protein.rdf file (Fig. 2) can be created by pressing Create. The RDF file can be customized by enabling Customize RDF File, which allows the addition of metals, cofactors, or water molecules and the determination of the protonation state of several amino acid residues (Fig. 3) ... 

Fig. 2. Creation of the receptor description file (RDF) for use with FlexX. Protein Structure Specify the correct PDB structure. Active-Site File The FlexX binding pocket can be defined as the amino acid residues within 7 A from a reference structure (e.g., a ligand structure translated to the protein active site midpoint as determined by PASS see Subheading 3.3.2.). Customize RDF File enables specification of metals, cofactors, protonation states, and torsions of residues (see Fig. 3).

Fig. 3. Customization of the receptor description file. Chains allows specification of the chains present in the protein that have to be included (grayed out if only one chain is present) Templates to add specific metals, cofactors, and/or water molecules or determine protonation state of Asp, Cys, Glu, His, Ser Torsions allows setting of the dihedral angle of the terminal hydrogen atom of the specified residue.

This is an auspicious time to publish a volume on copper proteins. The number of known proteins with metallic cofactors continues to increase steadily, and the availability of structural and sequence data is enabling much more specihc characterizations of the interactions between the metal ions and proteins as well as of their functions and mechanisms. Numerous investigators are choosing copper proteins and copper metabolism as their model systems for such studies. While copper-containing proteins play essential roles, their numbers are few enough that a comprehensive understanding is a reasonable goal. 

The simple coordination chemistry characteristic of the majority of protein-metal interactions is replaced in certain cases by irreversible covalent modifications of the protein mediated by the metal ion. These modifications are essential for the function and are templated by the structure of the protein, as no other proteins are required for the reaction to occur. These self-processing reactions result in the biogenesis of redox cofactors in some enzymes (amine oxidases, galactose oxidase, cytochrome c oxidase) and activation of hydrolytic sites in others (nitrile hydratase). The active sites of all of these enzymes are bifunctional, directing not only the catalytic turnover reaction of the mature enzyme but the modification steps required for maturation. 

Metallochaperones see Metallochaperones Metal Ion Homeostasis) are metal-binding proteins (Li) designed to deliver the appropriate metal ion or metal cofactor to a target ligand (L2), as illustrated in equation (2). 

Calcium-binding Proteins Copper Enzymes in Denitrification Copper Proteins with Type 1 Sites Copper Proteins with Type 2 Sites Iron Heme Proteins Electron Transport Iron-Sulfin Proteins Metal-mediated Protein Modification Metallochaperones Metal Ion Homeostasis Molybdenum MPT-containing Enzymes Nickel Enzymes Cofactors, Nitrogenase Catalysis Assembly Zinc Enzymes. 

Although the different sequences and folds of proteins provide the most disparate metal binding sites (some examples of which are provided in Fig. 3), Nature has evolved to select other organic or inorganic ligands for metal ions in proteins, which we call special metal cofactors. These cofactors can be grouped into two broad classes tetrapyrroles and metaUoclusters. 

Figure 3 Examples of metal cofactors in proteins (a) the zinc center of carbonic anhydrase, (b) the blue-copper center of plastocyanin, (c) the iron center in 2,3-dihydroxybiphenil dioxygenase, (d) the iron binding site of transferrin, and (e) the dinuclear copper site of Cu/ in cytochrome c oxidase.

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