AbstractThe microsomal (Mc) and mitochondrial (OM) isoforms of mammalian cytochrome b5are the products of different genes, which likely arose via duplication of a primordial gene and subsequent functional divergence. Despite sharing essentially identical folds, heme‐polypeptide interactions are stronger in OM b5s than in Mc b5s due to the presence of two conserved patches of hydrophobic amino acid side chains in the OM heme binding pockets. This is of fundamental interest in terms of understanding heme protein structure–function relationships, because stronger heme–polypeptide interactions in OM b5s in comparison to Mc b5s may represent a key source of their more negative reduction potentials. Herein we provide evidence that interactions amongst the amino acid side chains contributing to the hydrophobic patches in rat OM (rOM) b5persist when heme is removed, rendering the empty heme binding pocket of rOM apo‐b5more compact and less conformationally dynamic than that in bovine Mc (bMc) apo‐b5. This may contribute to the stronger heme binding by OM apo‐b5by reducing the entropic penalty associated with polypeptide folding. We also show that when bMc apo‐b5unfolds it adopts a structure that is more compact and contains greater nonrandom secondary structure content than unfolded rOM apo‐b5. We propose that a more robust β‐sheet in Mc apo‐b5s compensates for the absence of the hydrophobic packing interactions that stabilize the heme binding pocket in OM apo‐b5s.