Cobalt-substituted hemoprotein expression

Substituting the native metal of metalloenzymes can significantly alter the enzymes' reactivity and spectroscopic properties. Cobalt is especially attractive as a substitute for the native iron center in hemoproteins, as it generates metal variants with complementary spectroscopic properties and could enable new modes of reactivity. Here, we describe a detailed protocol for the biosynthesis and incorporation of cobalt protoporphyrin IX (CoPPIX) into hemoproteins, replacing the native heme b cofactor during expression in the common laboratory strain Escherichia coli BL21(DE3). This protocol is described using the model hemoprotein Physeter macrocephalus (sperm whale) myoglobin. Because of cobalt's unique electronic and geometric properties, cobalt-substituted hemoproteins offer a valuable handle for spectroscopic characterization and structural studies. We describe analytic methods of assessing cofactor identity and purity, including electronic absorption spectroscopy, liquid-chromatography/mass-spectrometry, inductively coupled plasma-mass spectrometry, and electron paramagnetic resonance spectroscopy. This method for generating artificial metalloenzymes is effective, easy to implement, and can produce useful quantities of Co-substituted hemoproteins.