Molecular Determinants of Efficient Cobalt-Substituted Hemoprotein Production in E. coli.

Exchanging the native iron of heme for other metals yields artificial metalloproteins with new properties for spectroscopic studies and biocatalysis. Recently, we reported a method for the biosynthesis and incorporation of a non-natural metallocofactor, cobalt protoporphyrin IX (CoPPIX), into hemoproteins using the common laboratory strain Escherichia coli BL21(DE3). This discovery inspired us to explore the determinants of metal specificity for metallocofactor biosynthesis in E. coli. Herein, we report detailed kinetic analysis of the ferrochelatase responsible for metal insertion, EcHemH (E. coli ferrochelatase). This enzyme exhibits a small, less than 2-fold preference for Fe2+ over the non-native Co2+ substrate in vitro. To test how mutations impact EcHemH, we used a surrogate metal specificity screen to identify variants with altered metal insertion preferences. This engineering process led to a variant with an ∼30-fold shift in specificity toward Co2+. When assayed in vivo, however, the impact of this mutation is small compared to the effects of alteration of the external metal concentrations. These data suggest that incorporation of cobalt into PPIX is enabled by the native promiscuity of EcHemH coupled with BL21's impaired ability to maintain transition-metal homeostasis. With this knowledge, we generated a method for CoPPIX production in rich media, which yields cobalt-substituted hemoproteins with >95% cofactor purity and yields comparable to standard expression protocols for the analogous native hemoproteins.