Metalloproteins Substitution

A detailed analysis of Ni11 complexes with mew-substituted porphyrins bearing zero, one, two, or four /-butyl groups revealed that both the out-of-plane and in-plane distortion depend on the perturbation symmetry of the peripheral substituents (number and position of substitutents), and their orientation.1775 These results have implications for understanding the role of nonplanar distortions in the function of metalloproteins containing nonplanar porphyrins.1776... 

In general, photolysis induces substitutional and redox-related changes, whereas pulse radiolysis primarily promotes redox chemistry. Indeed one of the unique features of the latter method is to induce unambiguous one electron reduction of multi-reducible centers. Metalloproteins can be rapidly reduced to metastable conformational states and subsequent changes monitored. 

The substitution process permeates the whole realm of coordination chemistry. It is frequently the first step in a redox reaction and in the dimerization or polymerization of a metal ion, the details of which in many cases are still rather scanty (e.g. for Cr(III) ). An understanding of the kinetics of substitution can be important for defining the best conditions for a preparative or analytical procedure. Substitution pervades the behavior of metal or metal-activated enzymes. The production of apoprotein (demetalloprotein and the regeneration of the protein, as well as the interaction of substrates and inhibitors with metalloproteins are important examples. ... 

Undoubtedly the most complicated mileau for a substitution process is that of a protein. However, the principles developed in this chapter for substitution in metal complexes also apply to metalloproteins. Allowance for a role for the protein, particularly near the site, must always be made. The formation and dissociation of a metalloprotein (PM) may be represented in an undoubtedly simplified form as ... 

Redox reactions usually lead, however, to a marked change in the species, as reactions 4-6 indicate. Important reactions involve the oxidation of organic and metalloprotein substrates (reactions 5 and 6) by oxidizing complex ions. Here the substrate often has ligand properties, and the first step in the overall process appears to be complex formation between the metal and substrate species. Redox reactions will often then be phenomenologically associated with substitution. After complex formation, the redox reaction can occur in a variety of ways, of which a direct intramolecular electron transfer within the adduct is the most obvious. 

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. 

Binuclear iron(II) complexes in which a hydroxide bridge is supported by the dinucleating bis-carboxylate ligand dibenzofuran-4,6-bis(diphenylacetate), (217), have proved useful models for hemerythrin. The nature of the binuclear iron center in hemerythrin itself, and in other metalloproteins, has been reviewed, the binding of O2, NO, N3, and NCS to the iron of hemerythrin discussed, " and the volume profile for hemerythrin reacting with O2 established. Bulky tolyl-substituted carboxylate ligands, both bridging and terminal, and... 

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...

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. 

The goal of many biomolecular NMR studies is characterization of global molecular structure. In metallo-biomolecules, and in particular, for paramagnetic species, it is sometimes preferable to use NMR to perform a more focused study of the metal ion coordination enviroiunent and the metal electronic structure. Metal sites show great variation in the effects on chemical shifts and line widths and thus often call for tailored approaches. In this section, characteristics of some of the metalloproteins metal sites most frequently studied by NMR are summarized. Examples have been selected to illustrate approaches described in this chapter such as metal substitution, use of pseudocontact shifts, RDCs, relaxation enhancement, and detection of nuclei other than H. 

Substitution of amino acid residues at the metal center by site-directed mutagenesis can alter the geometry and properties of the active site, thus allowing elucidation of the minimal requirements for the proper functioning of the metalloprotein. As an example. Figure 21 and Table 5 show the results of RR scattering from a series of P. aeruginosa azurins in which the active site Met 121 has... 

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