Characterization of de novo synthesized four-helix bundle proteins with metalloporphyrin cofactors

Two de novo synthesized proteins that form water-soluble four-helix bundle structures were constructed based on the modular synthesis strategy of template-assembled synthetic proteins. In the hydrophobic interior of these modular proteins one bis-histidine binding site was introduced to incorporate a single heme group or other metalloporphyrin cofactors. The exchange of two tryptophan against two arginine residues near the heme-binding site leads to variations in the stability of the modular proteins. The structural characterization of the de nova synthesized heme proteins by EPR spectroscopy revealed a heterogeneous binding situation of the cofactor in both systems. The heme group is coordinated by two axial histidine ligands, whose planes are oriented either parallel or twisted relative to each other. In one of the two modular proteins this heterogeneity was reduced by addition of guanidinium hydrochloride that aided in-the proper folding of the heme-binding site. By using different central metals for the protoporphyrin IX cofactor (Fe3+, Co3+, Zn2+) different functional properties were introduced into this modular protein. While the Fe3+ porphyrin is easily reduced, the reduction of Co3+ porphyrin is very difficult and finally leads to the release of this metalloporphyrin cofactor into the aqueous surrounding. By incorporating a porphyrin with a Zn2+ central metal a modular protein was obtained that was capable of acting as electron donor in light induced triplet electron transfer.