Metalloproteins histidine

Immobilized metal-affinity chromatography (IMAC) is also known as metal-chelate affinity chromatography (MCAC). This method was first proposed by Porath et al. in 1975 [63] and is based on the specific interactions between immobilized metal ions and amino acid residues, such as histidine, fiyptophan, and cysteine in proteins or peptides [63]. IMAC has become an important tool for the detection and purification of metalloproteins, histidine-tagged proteins, and phosphorylated proteins. Areas in which this method is now used include proteomics [64—66], work with recombinant proteins [67—69], and disease diagnosis [70,71]. 

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name). 

The normal cellular form of prion protein (PrPc) can exist as a Cu-metalloprotein in vivo (492). This PrPc is a precursor of the pathogenic protease-resistant form PrPsc, which is thought to cause scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt—Jakob disease. Two octa-repeats of PHGGGWGQ have been proposed as Cu(II) binding sites centered on histidine (493). They lack secondary and tertiary structure in the absence of Cu(II). Neurons may therefore have special mechanisms to regulate the distribution of copper. 

Carbonic anhydrase is a metalloprotein with a co-ordinate bonded zinc atom immobilized at three histidine residues (His 94, His 96 and Hisl 19) close to the active site of the enzyme. The catalytic activity of the different isoenzymes varies but cytosolic CA II is notable for its very high turnover number (Kcat) of approximately 1.5 million reactions per second. 

The complex [Ru(NH3)5H20] has proved to be a successful reagent for the modification of metalloproteins because of its affinity to accessible surface histidine residues. The Ru(NH3)j moiety has, for example, been attached to... 

Table 10. A comparison of rate constants for intramolecular electron transfer in Ru(NH3)j-modiiied electron transport metalloproteins, modifications at surface histidine present in native proteins, pH 7. Values of AE° by determined measurements on modified protein except as indicated...

As with any metalloprotein, the chemical and physical properties of the metal ion in cytochromes are determined by the both the primary and secondary coordination spheres (58-60). The primary coordination sphere has two components, the heme macrocycle and the axial ligands, which directly affect the bound metal ion. The pyrrole nitrogen donors of the heme macrocycle that are influenced by the substitutents on the heme periphery establish the base heme properties. These properties are directly modulated by the number and type of axial ligands derived from the protein amino acids. Typical heme proteins utilize histidine, methionine, tyrosinate, and cysteinate ligands to affect five or six coordination at the metal center. 

Our biomimetic investigations have focused on the metalloproteins hemocyanin (He) (11-17) and tyrosinase (11,12,14,16,18,29), which contain two copper ions in their active center. The function of hemocyanin is to bind and transport dioxygen in the hemolymph of molluscs and arthropods. Studies employing EXAFS spectroscopy have shown that in the deoxy form, two (19-21) or three (13,21) imidazole units fiom protein histidine residues coordinate to each cuprous ion. Upon addition of O2 to give oxy-Hc, considerable changes take place in the coordination sphere giving rise to tetragonally coordinated Cu(II) ions... 

Hemocyanin [30,31], tyrosinase [32] and catechol oxidase (2) [33] comprise this class of proteins. Their active sites are very similar and contain a dicopper core in which both Cu ions are ligated by three N-bound histidine residues. All three proteins are capable of binding dioxygen reversibly at ambient conditions. However, whereas hemocyanin is responsible for O2 transport in certain mollusks and arthropods, catechol oxidase and tyrosinase are enzymes that have vital catalytic functions in a variety of natural systems, namely the oxidation of phenolic substrates to catechols (Scheme 1) (tyrosinase) and the oxidation of catechols to o-quinones (tyrosinase and catechol oxidase). Antiferromagnetic coupling of the two Cu ions in the oxy state of these metalloproteins leads to ESR-silent behavior. Structural insight from X-ray crystallography is now available for all three enzymes, but details... 

Many metalloproteins contain more than one metal center. Tyrosinase, for example, has a dinuclear Type 3 (T3) copper active site which, in its oxidized form, comprises two Cu(II) centers each held by three histidine groups with a p-r 2 r 2 peroxido bridging ligand (Fig. 22). 

Many of the biologically active zinc metalloproteins contain a zinc(II) ion bound to one or more imidazole ligands of the amino add residue histidine. For this reason a large number of studies over an extended period have been carried out on zinc and cadmium complexes of imidazole, substituted imidazoles, histidine and related ligands. There has also been much recent activity in this field much structural information is available. 

Ru+2 complexes readily react with surface histidine residues to form stable derivatives. Photochemical methods were used to inject an electron into the Ru3+ site followed by monitoring kinetics of ET from Ru2+to the metalloprotein active site. 

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