Direct metalloproteins

Table 3.4 The main analytical techniques for direct metalloprotein analysis on electrophoresis gels.

The 2[4Fe-4S] and [3Fe-4S][4Fe-4S] ferredoxins are components of virtually all eubacteria and archaebacteria (3). Several comprehensive reviews dealing with these small metalloproteins have appeared (3, 8-12), but only those participating directly in the photosynthetic light reactions will be addressed here. 

Note, however, that the -59In mV change per pH-unit is seldom found for prosthetic groups in proteins because association of protons is usually not directly on the coordination complex (which could result in loss of the metal) but rather on a nearby (or not-so-nearby) amino-acid side chain. So, the change can be anywhere between 0 and -59In mV. This information can be quite valuable for an understanding of the mechanism of action of the metalloprotein, but it does mean that we have to carry out EPR-monitored redox titrations at several different pH-values. 

Some future directions in inorganic photochemistry have been outlined by Adamson (56). A pessimistic picture of the practical uses of solar energy conversion systems is painted, but a rosy view of the academic future of the subject is held. It is anticipated that there will be further examination of thermally equilibrated excited (thexi) states—their lifetimes, and spectroscopic and structural properties—and an extension of present efforts to organometallics and metalloproteins is also envisaged (56). The interpretation of spectroscopic data from excited states will continue to be controversial and require future experimentation (57). 

Objectives in the Application of Site-Directed Mutagenesis to the Study of Electron Transfer Metalloproteins... 

Table 1. Representative metalloproteins that bind redox-active metals and that have been studied by site-directed mutagenesis...

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. 

Direct electron transfer has also been achieved with many metalloproteins such as cytochrome C, horseradish peroxidase, microperoxidase (MP-11), myoglobin, hemoglobin, catalase, azurin, and so on, immobilized on different CNT-modified electrodes [45, 61, 144—153]. 

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. 

Fig. 5. (a and b) Two perpendicular orientations of direct carboxylate-zinc interactions retrieved from four metalloprotein structures contained in the Brookhaven Protein Data Bank. Orientation (a) represents the carboxylate group as found in Fig. 3. The coordination stereochemistry is syn for each example.

In contrast to n-n transitions, the n-n transitions of heterocyclic compounds and carbonyl-containing rings are often polarized in a direction perpendicular to the plane of the ring. Linear dichroism of cytosine, adenine, and other nucleic acid bases has been measured on single crystals and in partially oriented polymer films.71 Magnetically induced linear dichroism provides a new tool for study of metalloproteins.72... 

Zinc in metalloenzymes may (i) participate directly in the catalytic process, (ii) serve to stabilize protein structure or (iii) have a regulatory role. In each case, removal of the metal from the holoenzyme generally results in an apoprotein having no catalytic activity. The enzymes considered briefly below provide examples of each of these functions of Zn. The study of zinc metalloproteins has often in the past been beset by analytical problems and by contamination with traces of metal ions a review covering these important topics has appeared.1263 Another recent review deals with the physiological, nutritional and medical role of zinc.1264... 

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