Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins

De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 Å rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the GluXxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 Å. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation. P roteins use a limited repertoire of metal ion cofactors to help catalyze a multitude of reactions. For example, diiron sites (1-4) mediate reversible oxygen binding in hemerythrins, whereas they function as hydrolytic centers in phosphatases. Structurally similar diiron sites also mediate a number of oxygendependent oxidative processes. Ferritins serve as ferroxidases, while other diiron proteins catalyze hydroxylation, epoxidation, and desaturation reactions. Further, a diiron site in Escherichia coli ribonucleotide reductase is responsible for the formation of a Tyr radical. How do the structures of these proteins tune the chemical properties of a common diiron center to obtain such a diversity of highly specific catalysts? This question is being addressed through the study of the natural proteins as well as the study of small-molecule diiron complexes (1-4). Although impressive progress has been made on both fronts, these approaches have inherent limitations. The study of large proteins is hampered by their extreme complexity, and it is difficult to synthesize small-molecule models capable of simultaneously binding diiron, oxygen, and various substrates. Recently, we and others have sought a molecular middle ground between these two extremes through the design of small proteins and peptides that self assemble to form complexes with hemes and metal ions (5-8). Peptide models could have a number of distinct advantages relative to other synthetic models for metalloproteins. They allow the construction of hydrophobic pockets within watersoluble structures, and their structures may be synthesized easily and varied by using highly optimized methods of peptide synthesis. Further, peptide models address not only the issue of how the arrangement of atoms within an active site leads to function but also the very important question of how an amino acid sequence dictates the tertiary structure that supports this active site. Thus, several groups have described the design of minimal mimics of metalloproteins and heme-binding proteins that distill the quintessential elements believed to be responsible for the activities of metalloproteins into model proteins that are simpler and hence more easily understood than natural proteins (5-14). Through a careful characterization of the properties of such minimal metalloproteins, it should be possible to discern the features required for selective recognition of metal ions and for tuning their chemical properties. The protein-folding problem is a primary challenge encountered in the design of complex metalloproteins. This problem may be circumvented by grafting inorganic cofactor-binding sites into the structures of natural proteins that normally do not bind metal ions. Automated methods have been developed recently for engineering such ion-binding sites (15, 16), and it has been possible to build a number of structural as well as redox-active metal ion-binding sites within several different proteins However, complete control of a cofactor's environment might be best effected through the de novo design (27, 28) of proteins whose active sites are defined by the favorable free energy of folding of the polypeptide chain. Unfortunately, initial attempts to design proteins led to structures that formed molten globulelike states with dynamic behavior relative to natural proteins. More recently, it has been possible to design small uniquely folded proteins that incorporate all of the commonly occurring secondary structural and supersecondary structural motif