The 52-residue Desulfovibrio gigas rubredoxin peptide chain has been synthesized and a procedure for chain folding around iron(I1) developed. The folded, stable synthetic rubredoxin can be subjected to purification, and reversibly oxidized and reduced. Ultraviolet/visible absorption and CD spectra of both forms show all the same features as native D. gigas rubredoxin, and the symmetric and asymmetric Fe-S stretching bands in the resonance Raman spectrum can be identified. In addition, the matrix-assisted laser desorption mass spectrum of a peptide sample exposed to trace amounts of iron is dominated by a peak at 5735Da very close to the value for the calculated molecular mass. Details in the ultraviolethisible bandshape and mass spectrum, however, indicate remaining impurities. In comparison, a previously synthesized 25-residue rubredoxin fragment with the non-conserved positions 13-35 and 51 -52 omitted and Val5 -Glu50 anchored via glycine folds gives the correct molecular mass and ultraviolethisible spectrum, but is much more labile than the 52-residue protein. This shows that non-conserved residues are crucial in protein folding and that chemical metalloprotein synthesis offers alternative prospects to microbiological protein engineering.Total chemical synthesis of small, naturally occurring metalloproteins is becoming feasible, as indicated by recent studies of Clostridium pasteurianum ferredoxin [I], racemic Desulfovibrio desulfuricans rubredoxin [2], and horse heart cytochrome c [3]. While presently not competitive with microbiological methods, chemical metalloprotein synthesis is a potential powerful alternative to site-modification of proteins. These techniques can be used as tools for mapping physical and chemical properties of the protein, and factors important in maintaining the folded structure. Chemical metalloprotein synthesis also offers an additional perspective, namely that the peptide chain can both be modified and reduced in size. Metal complexes with peptide chain ligands large enough to fold but smaller than the chains in native proteins can thus be synthesized. This method would offer extremely efficient approaches to structural and functional metalloprotein mapping once the preparative procedures are powerful enough to provide the appropriate amounts of protein. The prospects are illustrated by a recently synthesized 61-residue protein with a novel three-strand p fold termed minibody [4]. Elements responsible for the stabilization due to folding for this protein, which forms a template for the heavy-chain domain of immunoglobin, were mapped and a binding site consisting of three histidine residues, following Correspondence to J. Ulstrup, Chemistry Department A, Building 207, The Technical University of Denmark, DK-2800 Lyngby, DenmarkAbbreviations. Fmoc , N-9-fluorenylmethox ycarbon yl ; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry. a metal-selectivity pattern similar to carbonic anhydrase, was engineered into the protein.Biomimetic metal complexes of peptides wi...