Metalloproteins copper

Polyphenol oxidase occurs within certain mammalian tissues as well as both lower (46,47) and higher (48-55) plants. In mammalian systems, the enzyme as tyrosinase (56) plays a significant role in melanin synthesis. The PPO complex of higher plants consists of a cresolase, a cate-cholase and a laccase. These copper metalloproteins catalyze the one and two electron oxidations of phenols to quinones at the expense of 02. Polyphenol oxidase also occurs in certain fungi where it is involved in the metabolism of certain tree-synthesized phenolic compounds that have been implicated in disease resistance, wound healing, and anti-nutrative modification of plant proteins to discourage herbivory (53,55). This protocol presents the Triton X-114-mediated solubilization of Vida faba chloroplast polyphenol oxidase as performed by Hutcheson and Buchanan (57). 

Studies with yeast cells have revealed that several proteins are used to pick up copper ions, as they are transported from the outside environment through the inside of the cell, and to shuttle them to appropriate copper metalloproteins. These shuttles, or "taxicabs," are proteins having names such as Atxl, Lys7, Cox 17, and CCC. Each of these proteins have a human equivalent. For example, the human equivalent of the yeast s CCC protein is the human Wilson s protein (Valentine and Gralla, 1997). 

Recently DFM, TFM and other methionine analogues were utilized to explore the contribution that methionine makes to the reduction-oxidation potential of the P. aeruginosa metalloprotein azurin [26, 45], Azurin is a copper metalloprotein that is involved in... 

An ever increasing number of copper metalloproteins is being recognized. Those regularly present in mammals are listed in Table 5 with some of their characteristics. Other important naturally occurring copper proteins, such as hemocyanin, laccase, and ascorbic acid oxidase, are not listed since they do not occur in mammals. The metalloprotein nature of some of the proteins listed in Table 5 has not been established fully as yet. The search for further copper proteins, copper-protein complexes, and other forms in which copper may be stored or transported in the body must continue. 

The copper(II) atoms present in copper metalloproteins have been classified according to their spectroscopic properties. Type-1, as found in blue copper proteins such as... 

The electron self-exchange rate constants for two Cu(I)/Cu(II) complex couples, synthesized as models for active sites of copper metalloproteins, have been determined in acetonitrile using NMR techniques. An upper limit of... 

The many redox reactions that take place within a cell make use of metalloproteins with a wide range of electron transfer potentials. To name just a few of their functions, these proteins play key roles in respiration, photosynthesis, and nitrogen fixation. Some of them simply shuttle electrons to or from enzymes that require electron transfer as part of their catalytic activity. In many other cases, a complex enzyme may incorporate its own electron transfer centers. There are three general categories of transition metal redox centers cytochromes, blue copper proteins, and iron-sulfur proteins. 

SOD comprises a family of metalloproteins primarily classified into four groups copper, zinc-containing SOD (Cu, Zn-SOD), manganese-containing SOD (Mn-SOD), iron-containing SOD (Fe-SOD) and nickel-containing SOD (Ni-SOD). In the following studies, we will only focus on the uses of the former three kinds of SODs to construct SOD-based 02 biosensors since the last one, Ni-SOD, is not commercially available. 

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

The transfer of a quadridentate N2S2-donor ligand from M2+ (M = Cr, Mn, Fe, Co or Ni) to Cu2+ (271), already mentioned in Section V.A.l, has a formal connection with an investigation of the mechanism of copper delivery to metalloproteins, such as copper zinc superoxide dismutase. Both are ligand exchange reactions of the type ML + CuL ML + CuL (300). 

Interest in this class of coordination compounds was sparked and fueled by the discovery that radical cofactors such as tyrosyl radicals play an important role in a rapidly growing number of metalloproteins. Thus, in 1972 Ehrenberg and Reichard (1) discovered that the R2 subunit of ribonucleotide reductase, a non-heme metal-loprotein, contains an uncoordinated, very stable tyrosyl radical in its active site. In contrast, Whittaker and Whittaker (2) showed that the active site of the copper containing enzyme galactose oxidase (GO) contains a radical cofactor where a Cu(II) ion is coordinated to a tyrosyl radical. 

Enzymes containing amino acid radicals are generally associated with transition metal ions—typically of iron, manganese, cobalt, or copper. In some instances, the metal is absent it is apparently replaced by redox-active organic cofactors such as S -adenosylmethionine or flavins. Functionally, their role is analogous to that of the metal ion in metalloproteins. 

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. 

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. 

As we will see in subsequent chapters, many metalloproteins have their metal centres located in organic cofactors (Lippard and Berg, 1994), such as the tetrapyrrole porphyrins and corrins, or in metal clusters, such as the Fe-S clusters in Fe-S proteins or the FeMo-cofactor of nitrogenase. Here we discuss briefly how metals are incorporated into porphyrins and corrins to form haem and other metallated tetrapyrroles, how Fe-S clusters are synthesized and how copper is inserted into superoxide dismutase. 

Electronic spectra of metalloproteins find their origins in (i) internal ligand absorption bands, such as n->n electronic transitions in porphyrins (ii) transitions associated entirely with metal orbitals (d-d transitions) (iii) charge-transfer bands between the ligand and the metal, such as the S ->Fe(II) and S ->Cu(II) charge-transfer bands seen in the optical spectra of Fe-S proteins and blue copper proteins, respectively. Figure 6.3a presents the characteristic spectrum of cytochrome c, one of the electron-transport haemoproteins of the mitochondrial... 

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