Many proteins bind metal ions, permanently as prosthetic groups or more transiently as ligands. These metal ions play a variety of roles in metalloproteins: electron transfer, maintaining the protein structure, oxygen binding, forming coordinated hydroxide radicals, substrate binding, and electrophilic catalysis (see Metal-Requiring Enzymes and Metalloproteinases).
During the past 15 years, it has become apparent that metalloproteins also play important roles in regulating the expression of genetic information. Such proteins take up stoichiometric quantities of trace metal ions and then undergo conformational changes that produce marked differences in their abilities to bind to specific sites on the genomic DNA or RNA of a given organism. The physiological effects of the binding of metalloproteins to, or release from, nucleic acids include increased resistance to heavy metals, control of iron uptake and storage pathways, recognition of packaging signals within the RNAs of retroviruses, control of vertebrate development events, andrecognition of steroid hormones. Minerals present in high concentrations, such as Na+, K+, and 2+Mg , can play important roles in stabilizing nucleic acid structures, but they are unlikely to be widely used in gene regulation. The remaining essential metals are normally trace elements. Hence, it is not surprising that zinc, iron, and copper are prominent metalloregulators (see also Calcium-Binding Proteins, Zinc-Binding Proteins, and Iron-Binding Proteins). The incorporation of a particular trace metal ion into an apoprotein is influenced by its ionic radius, thermodynamic stability, ligand-substitution kinetics, and charge (Table 1). Three to four ligands, usually the side chains of cysteine and histidine residues, typically complete the coordination sphere about a bound metal ion.
Table 1. Coordination Environments Preferred by Common Metal Ions Found in Proteins
|
Metal Ion Coordination Number |
Geometry |
Ligands |
|
 |
4 |
Tetrahedral |
His, Cys |
 |
4 |
Tetrahedral His, |
Cys, Glu, Asp |
 |
4 |
Tetrahedral |
Cys |
 |
4 |
Tetrahedral |
Cys |
|
Hg2+ |
3 |
Trigonal |
Cys |
Because of their intrinsic biological functions, regulatory metalloproteins are not normally present in large quantities in cell and tissue homogenates, and most are colorless or only weakly colored. Nonetheless, the study of regulatory metalloproteins has become the fastest growth area of inorganic biochemistry. The term "metalloregulation" refers to regulating the transfer of genetic information by metal ions.
Regulatory metal-binding proteins are generally specific for a particular trace metal ion. For example, a synthetic zinc finger analogue (1) displays a strong preference for
1. Characterization
As with any metalloprotein, the metal stoichiometry is of paramount importance. It is usually determined by titration or atomic absorption spectrometry. Several thousand putative metalloregulatory proteins have been reported in the literature, usually on the basis of limited sequence patterns alone. In the absence any physical characterization, however, such reports must be viewed as speculative because they offer no real evidence regarding the metal element involved.
The nature of the metal coordination sphere can be probed by electronic absorption spectroscopy incases involving iron or copper coordination. Zn presents a challenge because it is colorless, but the2+ 2+ Zn can be replaced with Co , a chromophoric probe that possesses a comparable ionic radius, is kinetically labile, and prefers similar coordination environments.
X-ray absorption spectroscopy is particularly useful for investigating the local environment (<~5) of the metals in these proteins (2). When X rays are absorbed by metal atoms, they liberate electrons that are backscattered from neighboring (ie, ligand) atoms. This interference phenomenon provides a very precise measurement of the distance to neighboring atoms. The edge region of the spectrum provides information about the valence state and coordination geometry of the metal and the identity of neighboring atoms. The extended X-ray absorption fine structure (EXAFS) region of the spectrum contains information about the number and average distances of neighboring atoms and about their relative disorder (see EXAFS).
The structural methods do not address key issues regarding the way a regulatory protein acts as an information transducer. Electrophoresisexperiments, including hydroxyl-radical footprinting and mobility shift assays, can indicate what region of the DNA or RNA is recognized and whether protein binding induces a structural change in the nucleic acid. More precise structural information relating to the nature of the protein-nucleic acid contacts requires their cocrystallization and X-ray crystallographic structural determination. Solution nuclear magnetic resonance (NMR) is also used in structural determination of small metalloproteins, but it has not yet been used to study complexes with DNA or RNA. Approximately 50 structures of metalloproteins have been deposited to date in the Brookhaven Protein Data Bank (see Structure Databases).
2. Regulatory Metalloproteins
The major regulatory metalloproteins are described in Zinc-binding proteins, Zinc fingers, Iron-binding proteins, Iron-response elements, and Metal-response elements. In addition, organisms must frequently cope with toxic elements in the environment. Bacteria are especially rich sources of metalloregulators and encode resistance systems (3) for many toxic metal ions, including
Heavy-metal resistance is achieved in various ways, including sequestration in proteins, such as metallothioneins, efflux "pumping," and reduction to volatile metal atoms (eg, Hg0).
3. Concluding Remarks
In contrast to the surging interest in structural studies on metalloproteins, the metabolic effects of dietary metal supply on the availability of metalloregulatory proteins have not received nearly as much attention as is warranted. Sorting out these effects is complicated by the presence of trace metal ions in structural proteins and enzymes. For example, zinc is found in RNA polymerase and in zinc fingers.
The sensing of small molecules by metalloproteins is only beginning to be understood. How are gases of physiological relevance (eg, O2, CO, NO, CO2) recognized by proteins other than the heme- containing oxygen-binding proteins and cytochrome oxidases, and what are the regulatory consequences at the genetic level? For example, the requirement for CO 2 as an environmental determinant of gene expression involved in many bacterial capsule and toxin biosyntheses has long been recognized by microbiologists but is not understood. Curiously, heme proteins (4), rather than the nonheme iron-responsive proteins discussed in Iron-response elements, have been implicated in most of what is presently known about the molecular mechanisms of gas sensing in biology.
The initial reports of mutant metal-binding proteins have naturally led to their consideration for use in the biotechnology industry. The development of artificial regulators of eukaryotic gene expression is a much sought after goal. Can metalloprotein-based drugs be produced that block the expression of genes whose products are important in the developing animal and plant diseases? Alternatively, could such types of regulatory proteins be used to produce large transgenic animals and plants? Novel zinc-finger proteins designed to function as specific transcriptional switches have been reported (5, 6). Other potential applications for engineered metal-binding proteins include highly sensitive metal-ion biosensors.
