De novo protein design as a methodology for synthetic bioinorganic chemistry.

The major advances in molecular and structural biology and automated peptide and DNA synthesis of the 1970s and 1980s generated fertile conditions in the 1990s for the exploration of designed proteins as a new approach for inorganic chemists to generate biomolecular mimics of metalloproteins. This Account follows the development of the TRI peptide family of three-stranded coiled coils (3SCC) and α3D family of three-helix bundles (3HB) as scaffolds for the preparation of metal binding sites within de novo designed constructs. The 3SCC were developed using the concept of a heptad repeat (abcdefg) putting hydrophobes in the a and d positions. The TRI peptides contain four heptads with capping glycines. Via substitution of leucine hydrophobes, metal ligands can be introduced into the a or d sites in order to bind metals. First, the ability to use cysteine-substituted 3SCC aggregates to impose higher or lower coordination numbers on Hg(II) and Cd(II) or matching the coordination preferences of As(III) and Pb(II) is discussed. Then, methods to develop dual site peptides capable of discriminating metals based on their type (e.g., Cd(II) vs Pb(II)), their preference for a vs d sites, and then their coordination number is described. Once these principles of metal site differentiation are described, we shift to building dual site peptides using both cysteine and histidine metal binding sites. This approach provides a construct with both a Hg(II) structural and a Zn(II) hydrolytic center, the latter of which is capable of hydrating CO2. With these Zn(II) proteins, we consider the relative importance of the location of the catalytic center along the primary sequence of the peptide and show that only minor perturbations in catalytic efficiencies are observed based on metal location. We then assess the feasibility of preparing enzymes competent to reduce nitrite with copper centers in a histidine-rich environment. As part of this discussion, we examine the influence of surface residues on catalyst reduction potentials and catalytic efficiencies. We end describing approaches to prepare asymmetric proteins that can incorporate acid-base catalysts or water channels. In this respect, we highlight modifications of a helix-turn-helix-turn-helix motif called α3D and show how this 3HB can be modified to bind heavy metals or to make Zn(II) centers, which are active hydrolytic catalysts. A comparison is made to the comparable parallel 3SCC.

Substituted imidazole of 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates cytochrome P450 2D6 by protein adduction.

5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) is a potent mechanism-based inactivator of human cytochrome P450 2D6 that displays type I binding spectra with a K(s) of 0.39 ± 0.10 µM. The partition ratio is ~3, indicating potent inactivation that addition of exogenous nucleophiles does not prevent. Within 15 min of incubation with SCH 66712 and NADPH, ∼90% of CYP2D6 activity is lost with only ~20% loss in ability to bind CO and ~25% loss of native heme over the same time. The stoichiometry of binding to the protein was 1.2:1. SDS-polyacrylamide gel electrophoresis with Western blotting and autoradiography analyses of CYP2D6 after incubations with radiolabeled SCH 66712 further support the presence of a protein adduct. Metabolites of SCH 66712 detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron.