Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues

Abstract

Conspectus Heme proteins have proven to be a convenient platform for the development of designer proteins with novel functionalities. This is achieved by substituting the native iron porphyrin cofactor with a heme analogue that possesses the desired properties. Replacing the iron center of the porphyrin with another metal provides one inroad to novel protein function. A less explored approach is substitution of the porphyrin cofactor with an alternative tetrapyrrole macrocycle or a related ligand. In general, these ligands exhibit chemical properties and reactivity that are distinct from those of porphyrins. While these techniques have most prominently been utilized to develop artificial metalloenzymes, there are many other applications of this methodology to problems in biochemistry, health, and medicine. Incorporation of synthetic cofactors into protein environments represents a facile way to impart water solubility and biocompatibility. It circumvents the laborious synthesis of water-soluble cofactors, which often introduces substantial charge that leads to undesired bioaccumulation. To this end, the incorporation of unnatural cofactors in heme proteins has enabled the development of designer proteins as optical oxygen sensors, MRI contrast agents, spectroscopic probes, tools to interrogate protein function, antibiotics, and fluorescent proteins. Incorporation of an artificial cofactor is frequently accomplished by denaturing the holoprotein with removal of the heme; the refolded apoprotein is then reconstituted with the artificial cofactor. This process often results in substantial protein loss and does not necessarily guarantee that the refolded protein adopts the native structure. To circumvent these issues, our laboratory has pioneered the use of the RP523 strain of E. coli to incorporate artificial cofactors into heme proteins using expression-based methods. This strain lacks the ability to biosynthesize heme, and the bacterial cell wall is permeable to heme and related molecules. In this way, heme analogues supplemented in the growth medium are incorporated into heme proteins. This approach can also be leveraged for the direct expression of the apoprotein for subsequent reconstitution. These methodologies have been exploited to incorporate non-native cofactors into heme proteins that are resistant to harsh environmental conditions: the heme nitric oxide/oxygen binding protein (H-NOX) from Caldanaerobacter subterraneus (Cs) and the heme acquisition system protein A (HasA) from Pseudomonas aeruginosa (Pa). The exceptional stability of these proteins makes them ideal scaffolds for biomedical applications. Optical oxygen sensing has been accomplished using a phosphorescent ruthenium porphyrin as the artificial heme cofactor. Paramagnetic manganese and gadolinium porphyrins yield high-relaxivity, protein-based MRI contrast agents. A fluorescent phosphorus corrole serves as a heme analogue to produce fluorescent proteins. Iron complexes of nonporphyrin cofactors bound to HasA inhibit the growth of pathogenic b...

Corrole–protein interactions in H-NOX and HasA

“…Haem proteins represent a versatile platform for the development of designer proteins with novel functionality, [1][2][3] most prominently as artificial metalloenzymes. Often, haem proteins are directly used with the native iron porphyrin cofactor.…”

Ratiometric Oxygen Sensing with H-NOX Protein Conjugates

“…RP523 cells lack the ability to biosynthesize heme and the cell wall is permeable to heme and related analogs. When grown anaerobically, these bacteria readily incorporate heme analogs supplemented in the growth medium into heme proteins . Both WT and S84C Cs H-NOX were grown on an ∼7 L scale and supplemented with ∼50 mg of Pd­(MPIX).…”

“…This protein is extremely stable, is readily expressed in Escherichia coli, is amenable to mutagenesis, and binds unnatural heme cofactors. Together, these properties render Cs H-NOX an ideal platform to construct novel sensing and imaging agents. , Consequently, Cs H-NOX has been tailored for a variety of applications, including nitric oxide sensing, MRI imaging, fluorescent labeling, and fluorescent proteins . We have previously reported protein-based optical oxygen sensors where the native heme cofactor in myoglobin or Cs H-NOX was substituted with the Ru­(II)–CO complex of mesoporphyrin IX (MPIX) as the O 2 -responsive phosphor .…”

“…When grown anaerobically, these bacteria readily incorporate heme analogs supplemented in the growth medium into heme proteins. 31 Both WT and S84C Cs H-NOX were grown on an ∼7 L scale and supplemented with ∼50 mg of Pd(MPIX). The protein exhibited a high degree of porphyrin incorporation, but not as much as the protein reconstituted with Pd(MPIX).…”

TEtraQuinolines (TEQs): A missing link in the family of porphyrinoid macrocycles

“…Porphyrin, a nitrogen-functionalized pseudo-D4h-symmetric 18π aromatic macrocycle, is arguably one of the most important naturally occurring small cyclic molecules, whose metal complexes enable a myriad of enzymatic chemical transformations as essential cofactors and serve as key signalling and transportation substances in a vast array of living organisms 1,2 . Thus, porphyrins have inspired decades of intense research on their structural modification, which has led to a variety of porphyrinoids and their metal complexes in order to explore new molecular functions [3][4][5][6][7] . Among the related macrocycles that feature four inwardly oriented nitrogen atoms, structures are usually characterized according to the number of protonated and positively charged nitrogen atoms and the overall architecture (Fig.…”