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.