The direct expression of apo heme proteins: A general strategy to obtain designer heme proteins with abiological cofactors.

Substituting the porphyrin cofactor in heme proteins with non-iron porphyrins or related tetrapyrrole macrocycles provides one strategy to obtain new functional proteins. This approach commonly relies on the removal of heme from the native holoprotein under harsh, denaturing conditions, which is not suitable for all proteins. To circumvent this limitation, the RP523 strain of E. coli can be utilized for the expression-based incorporation of abiological cofactors by leveraging the unique heme permeability of this strain's cell wall. However, not all synthetic analogs are able to traverse the RP523 cell wall. Here, we report the direct expression of apo heme proteins, which can then be reconstituted with the desired cofactor, thereby expanding the utility of the RP523 approach. As a test case, we describe the incorporation of zinc protoporphyrin IX (PPIX) into the heme nitric oxide/oxygen binding protein (H-NOX) from Caldanaerobacter subterraneus (Cs) and the heme acquisition system protein A (HasA) from Pseudomonas aeruginosa (Pa). Since the optical properties of the cofactor are sensitive to subtle differences in the protein environment, we compare the absorption and emission spectroscopy of these conjugates. It was found that higher cofactor incorporation was achieved by reconstitution. However, higher quantum yields were observed for samples prepared by expression-based methods, suggesting greater fidelity in cofactor binding. Our method for the direct expression of apo heme proteins substantially reduces the required amount of the cofactor and represents a generalizable approach for the incorporation of porphyrin analogs into heme proteins.