Natural enzymes exhibit high catalytic efficiency and selectivity for their dedicated chemical transformations, and the power of enzymes have enticed chemists to scrutinize the chemical mechanisms of their catalytic cycles. From the studies of a carboxylate-bridged diiron enzyme, toluene/o-xylene monooxygenase hydroxylase, we found that metal ions generate catalytically active intermediates, and the protein environment is more than a site for catalysis as a sophisticatedly programmed matrix that regulates overall processes. Inspired by the beauty of the biochemical architecture orchestrated with metal ions, we have designed a protein that self-assembles into a tetramer via interactions of hydrophobic residues, formation of disulfide bonds, and coordination of metal ions. The protein also creates catalytic zinc sites, which can be further optimized for selective interaction with a substrate, ampicillin, by directed evolution. We have learned that design of protein-based biocatalysts can be considered as a distinctive route of reproducing and developing orchestrated chemical/biological events. Furthermore, we suppose that protein engineering can aid us to develop efficient and sustainable biocatalysts and biomaterials in conjunction with synthetic biology, bioinformatics, and other related fields.