Incorporation of modified and artificial cofactors into naturally occurring protein scaffolds

As a possible modification of cofactor-containing proteins, cofactor-substitution typically leads to drastic changes of protein function. In particular heme, a porphyrin iron complex, is a representative, replaceable cofactor for this methodology and numerous cofactor-modified hemoproteins (reconstituted hemoproteins) have been prepared with the goal of elucidating their operational mechanism and/or engineering the protein function. In a series of hemoproteins, myoglobin, an oxygen storage hemoprotein, is one of the most rewarding scaffolds to generate a modified protein with an artificial cofactor. In this chapter, we describe practical procedures for the preparation of apomyoglobin and incorporation of zinc porphyrin as an artificial cofactor. Furthermore, we discuss the methodology to characterize the obtained cofactor-substituted proteins and the design of several artificial cofactors.