Functionalization of Bacterial Microcompartment Shell Proteins With Covalently Attached Heme

Area of Science:

• Biochemistry and Synthetic Biology
• Bioelectronics and Biomaterials

Background:

• Heme is a crucial redox cofactor with significant potential in synthetic biology and bioelectronics.
• Attaching heme to non-heme-binding proteins in vivo could broaden bioelectronic material diversity, especially for position-sensitive electron transfer.

Purpose of the Study:

• To develop a straightforward in vivo method for incorporating hemes into protein backbones using the cytochrome maturation system I.
• To functionalize self-assembling bacterial microcompartment shell proteins with hemes at defined locations.

Main Methods:

• Utilized the cytochrome maturation system I for in vivo heme incorporation.
• Targeted bacterial microcompartment shell proteins, introducing functional hemes into the protein backbone via amino acid substitutions.
• Employed spectroscopic measurements to characterize heme binding and redox properties.

Main Results:

• Successfully incorporated hemes into target proteins with high occupancy by substituting three amino acids.
• Spectroscopic data confirmed covalent binding of low-spin hemes with redox midpoint potentials around -210 mV vs. SHE.
• Heme-modified proteins partially retained self-assembly capabilities (hexamerization, inter-hexamer attachments) and integrated into higher-order structures, though with some alterations.

Conclusions:

• A versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins in vivo has been established.
• Provides insights into the interplay between heme incorporation and native protein assembly properties for engineered proteins.