The X-ray crystallographic structure of a designed, self-assembling, and heme-binding protein

De novo heme-binding proteins offer powerful minimal scaffolds for elucidating how sequence, oligomerization, and environment rule cofactor coordination, yet high-resolution structural data for these systems remain scarce. Here, we report the high-resolution crystal structure of a designed, self-assembling α-helical and heme-binding protein determined at 1.9 Å resolution, that undergoes a pronounced pH- and cofactor-driven reorganization. The structure crystallized in the orthorhombic P2₁2₁2₁ space group (a = 44.8 Å, b = 46.3 Å, c = 101.2 Å), with one tetramer per asymmetric unit and refinement statistics of R_fac/R_free = 23.5/25.5%. The apo protein folds into a three-stranded coiled coil, whereas heme binding under strongly basic conditions disrupts register-specific interactions and drives a major helix shift. This transition yields a four-stranded coiled coil with an out-of-register ABAB arrangement that accommodates two bis-histidine-ligated hemes. Cofactors are His-coordinated via the ε-nitrogen (Fe-N_His ≈ 2.0 Å), separated by 38 Å (tetrapyrroles edge-to-edge) and arranged nearly coplanar with a 67° rotation offset relative to one another. Imidazole dihedral angles (99° and 108°) indicate near-orthogonal geometry for the His axial ligands to the heme. Crystallization is promoted by a heme-templated packing in which interactions between macrocycles of neighboring assemblies and several salt-bridges generate an extended supramolecular lattice. These results demonstrate how heme binding and proton concentration reshape oligomeric topology and promote ordered assembly in minimal protein systems, highlighting the varied structural effects of heme binding and providing principles for stabilizing specific coordination and exploiting cofactor-cofactor interactions to guide high-order organization in de novo metalloproteins.