Manabu Ishida

Design of a Novel Heme Protein with a Non-Heme Globin Scaffold

A binding site for iron protoporphyrin IX (heme) was designed and embedded in a photosynthetic non-heme protein, phycocyanin, which forms a globin-like backbone structure, called a globin fold, but lacks sequence similarity to the globin family containing myoglobins and hemoglobins. Based on the structural alignment of the phycocyanin and myoglobin molecules, the proximal and distal His residues were repositioned in the phycocyanin sequence for heme ligation. The heme-binding pocket was created around the His residues by several residue replacements in the phycocyanin core. The synthesized phycocyanin variant, designated as HPY, bound one heme per protein molecule and showed spectroscopic features characteristic of six-coordinated heme proteins. The heme-binding HPY exhibited redox activity with an electrochemical midpoint potential of -130 mV against the standard hydrogen electrode, which was approximately 200 mV lower than the potential of natural myoglobins but 50 mV higher than the typical values of designed heme proteins with four-helix bundle or globin scaffolds. HPY also displayed native-like folding properties, in contrast to these designed heme proteins. However, the bound ferrous heme of HPY was quickly reoxidized by air and did not stably bind O(2), unlike the natural globins. The present results demonstrated that the globin fold of a non-globin protein is suitable for binding heme but is not sufficient for the reversible O(2) binding and myoglobin functions. The comparison of HPY with the natural globins may yield new insights into the essential features for realizing the natural heme protein functions.

Structural and functional properties of designed globins

De novo design of artificial proteins is an essential approach to elucidate the principles of protein architecture and to understand specific functions of natural proteins and also to yield novel molecules for medical and industrial aims. We have designed artificial sequences of 153 amino acids to fit the main-chain framework of the sperm whale myoglobin structure based on the knowledge-based energy functions to evaluate the compatibility between protein tertiary structures and amino acid sequences. The synthesized artificial globins bind a single heme per protein molecule as designed, which show well-defined electrochemical and spectroscopic features characteristic of proteins with a low-spin heme. Redox and ligand binding reactions of the artificial heme proteins were investigated and these heme-related functions were found to vary with their structural uniqueness. Relationships between the structural and functional properties are discussed.