Composite metalloproteins

In this chapter, we overview first some recent examples of interfacial electrochemical ET of composite metalloproteins where molecular mechanistic detail has in some way been achieved. We discuss next some theoretical issues regarding in situ STM of large molecules, where resonance or environmentally activated tunnel channels are opened by the redox metal centre. This is followed by an overview of some recent achievements in the area of in situ STM/AFM of the single-metal proteins cytochrome c and azurin on polycrystalline and single-crystal platinum and gold surfaces. Such an integrated approach offers new perspectives for experimental and theoretical characterization of metalloproteins at solid surfaces in contact with the natural aqueous medium for metalloprotein function. 

The rate constants also hold a useful clue to concentration and time ranges for new investigations of intermolecular ET processes of cyt c4 in solution, from which the intramolecular rate constants may be substantiated. Voltammetry, therefore, stands forward as a powerful tool towards otherwise elusive ET dynamics in composite metalloproteins. 

Investigations of multicentre electrochemical metalloprotein function including metalloenzyme function have also been brought to a level, where both direct and catalytic modes, and elements of molecular mechanisms can be addressed. The latter are, however, entangled by features such as composite electrochemistry, extremely complicated molecular interaction patterns when more than two metallic redox centres are involved, fragile surface enzyme preparations, and lack of structural surface characterization of the adsorbed metalloenzymes. In this respect, two-centre metalloproteins constitute interesting promising intermediates where the coop-erativity between the metallic redox centres can be accurately addressed with molecular resolution within reach. 

Investigations of protein-protein ET reactions have provided important insights into biological electron flow [10-14]. Natural systems, however, often are not amenable to the systematic studies that are required for evaluation of the key ET parameters 2 and Nab- A successful alternative approach involves measurements of ET in metalloproteins that have been labeled with redox-active molecules [15-19]. By varying the binding site and chemical composition of the probe molecule, it has been possible to elucidate the factors that control the rates of long-range ET reactions in proteins. 

The most definitive assessment of the metal composition of metalloproteins comes from the application of element-specific detection methods. CE-ICP-MS provides information not only about the type and quantity of individual metals bound to the proteins but also about the isotopes of each element as well [11,12]. Elemental speciation has become increasingly important to the areas of toxicology and environmental chemistry. Such analytical capability also opens up important possibilities for trace element metabolism studies. Figure 1 depicts the separation of rabbit liver metallothionein containing zinc, copper, and cadmium (the predominant metal) using CE-ICP-MS with a high-sensitivity, direct injection nebulizer (DIN) interface. UV detection (200 nm) was used to monitor the efficiency of the CE separation of the protein isoforms (MT-1 and MT-2), whereas ICP-MS detection made it possible to detect and quantify specific zinc, copper (not shown), and cadmium isotopes associated with the individual isoform peaks. 

With few exceptions, metallothioneins consist of relatively simple amino acids, aromatic amino acids and histidine only being found in a small number of species [329]. This amino acid composition suggests that metallothioneins evolved early in the evolution of life, probably even before the oxygenation of the atmosphere. A further clue is one of their functions. As metal-transport and storage proteins, thioneins are capable of binding metal ions but release them relatively easily as well. Metallothioneins can therefore be considered a transition from non-metal to metalloproteins. It is improbable, however, that the known copper proteins evolved from copper metallothioneins as there are no homologies between them and other copper proteins or enzymes. 

Vallee BLandWACKERWEC (1970) Metalloproteins. In Neurath H and Hill R, eds. The Proteins Composition, Structure and Function, Vol. 5, pp. 1-192. Academic Press, New York. 

In this chapter, the key topics of artificial metalloproteins utilizing protein vacant space are reviewed, outhning recent studies since 2000. Section 3 shows the approaches for construction of the metal complex/protein composites and their crystal structures and, further, describes the interactions between metal-drugs and proteins. Section 4... 

For the construction of artificial metalloproteins, protein scaffolds should be stable, both over a wide range of pH and organic solvents, and at high temperature. In addition, crystal structures of protein scaffolds are crucial for their rational design. The proteins reported so far for the conjugation of metal complexes are listed in Fig. 1. Lysozyme (Ly) is a small enzyme that catalyzes hydrolysis of polysaccharides and is well known as a protein easily crystallized (Fig. la). Thus, lysozyme has been used as a model protein for studying interactions between metal compounds and proteins [13,14,42,43]. For example, [Ru(p-cymene)] L [Mn(CO)3l, and cisplatin are regiospecificaUy coordinated to the N = atom of His 15 in hen egg white lysozyme [14, 42, 43]. Serum albumin (SA) is one of the most abundant blood proteins, and exhibits an ability to accommodate a variety of hydrophobic compounds such as fatty acids, bilirubin, and hemin (Fig. lb). Thus, SA has been used to bind several metal complexes such as Rh(acac)(CO)2, Fe- and Mn-corroles, and Cu-phthalocyanine and the composites applied to asymmetric catalytic reactions [20, 28-30]. 

Once electroactivity has been achieved for a metalloprotein, the voltammetric waves become a valuable signature of the active site(s). Voltammetry can therefore be used to define states and to monitor changes in structure and composition that may result from appropriate chemical or electrochemical perturbations of the system. This has interesting and important consequences since an electrochemical experiment provides conditions of strict potential control within a small and often microscopic sample size. The applications are, at present, in their infancy, but I shall endeavour to illustrate the opportunities for studies of metal site speciation by reference to one topical area, that of metal-ion uptake and loss among Fe-S clusters. 

The most definitive assessment of the metal composition of metalloproteins comes from the application of element-specific detection methods. CE/ICP-MS provides information not only about the type and quantity of individual metals bound to the proteins but also about the isotopes of each element as well. Elemental speciation has become increasingly important to the areas of toxicology and environmental chemistry. Such analytical capability also opens up important possibilities for trace element metabolism studies. Fig. 1 depicts... 

In addition to the monograph on the iron-sulphur proteins, there have been two reviews on this important class of macromolecule, and the lUPAC-IUB commission on biochemical nomenclature has published recommendations on their nomenclature. A method has been described for the interpretation of e.p.r. spectra of reduced dinuclear iron-sulphur proteins which will allow both the symmetry and the extent of covalency at the paramagnetic site to be parameterized. The parameters can then be related to the chemical composition of the paramagnetic centre, the protein-dependent charge delocalization of the unpaired electron, and the geometrical arrangement at the reduced iron atom - an analysis which, it is hoped, will ultimately be useful in rationalizing the redox behaviour of these important metalloproteins. 

Metalloproteomics is a new subject focusing on the distributions and compositions of all metalloproteins in a proteome (metalloproteome), their structural and functional characterization and their structural metal binding moieties. The specificity of metalloproteomics studies demands the need for a description of the metal-binding sites, metal stoichiometry, and metal-depen-dent structure or conformation changes as well as the identifieation and quantification of the metalloproteins. The metalloproteome can be considered as not only a subset of the metallome, but also a very important subset of the proteome. 

The study focusing on the distributions and 31,34 compositions of all metalloproteins in a proteome, their structural and functional characterization and their structural metal binding moieties... 

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