Design and preparation of artificial heme-copper enzymes

Natural and artificial metalloproteins play a critical role in biochemistry, with the first X-ray crystal structures ever solved belonging to heme proteins Due to their ability to carry out a diverse array of challenging reactions at ambient temperature, effective metalloenzyme design and isolation strategies are highly desirable. Control of active site geometry is often the key requirement for catalysis and its mutagenesis helps probe a wide variety of biological and abiological reactions. In the case of small-molecule activation, introduction of new metal-binding sites to non-native heme scaffolds can unlock new chemistry. In this chapter, we will provide methods used in our lab for the design and experimental preparation of artificial metalloenzymes containing a heme-copper center to mimic and understand heme-copper oxidases. The methods can be applied to design other heterobinuclear centers containing heme, such as the heme-nonheme iron center in nitric oxidase reductases.