To investigate the properties of unnatural zinc finger peptides with CysHis(3)-type ligand combinations, the HCHH- and CHHH-type zinc finger proteins (zf(HCHH) and zf(CHHH), respectively) were created by mutating Cys to His in the Cys(2)His(2)-type zinc finger of the transcription factor Spl (zf(CCHH)). The CD measurements clearly show that the single-finger CysHis(3)-type zinc finger peptides (zf(HCHH)f2 and zf(CHHH)f2) are folded by complexation with Zn(II). From the gel mobility shift assays, the CysHis(3-)type zinc finger proteins (zf(HCHH) and zf(CHHH)) evidently bind to the GC-box DNA, though the DNA binding affinity is lower than that of the wild CCHH-type zinc finger protein. Furthermore, the zf(HCHH)f2 and zf(CHHH)f2 peptides catalyze the hydrolysis of the 4-nitrophenyl acetate in contrast to the catalytically inactive zf(CCHH) peptide. This is the first study of the CysHis(3)-type zinc finger proteins and also provides useful information for redesigning artificial metalloproteins. (C) 2004 Elsevier Inc. All rights reserved.