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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. 

The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector‐driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA. We employed site‐directed spin labeling electron paramagnetic resonance spectroscopy to probe CooA motions on the μs‐ms timescale. We created a series of Cys substitution variants, each with a cysteine residue introduced into a key functional region of the protein: K26C, E60C, F132C, D134C, and S175C. The heme environment and DNA binding affinity of each variant were comparable to those of wild‐type CooA, with the exception of F132C, which displayed reduced DNA binding affinity. This observation confirms a previously hypothesized role for Phe¹³²in transmitting the allosteric CO binding signal. Osmolyte perturbation studies of Fe(III) “locked‐off” CooA variants labeled with either MTSL or MAL‐6 nitroxide spin labels revealed that multicomponent EPR spectra report on conformational flexibility on the μs‐ms timescale. Multiple dynamic populations exist at every site examined in the structurally uncharacterized Fe(III) “locked‐off” CooA. This observation suggests that, in direct contrast to effector‐free CRP, Fe(III) “locked‐off” CooA undergoes conformational exchange on the μs‐ms timescale. Importantly, we establish MAL‐6 as a spin label with a redox‐stable linkage that may be utilized to compare conformational dynamics between functional states of CooA.