Abstract Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user‐defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron‐conducting circuitry. Here, we use a set of de novo designed mono‐heme and di‐heme soluble and membrane proteins to delineate the contributions of electrostatic micro‐environments and dielectric properties of the surrounding protein medium on the inter‐heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water‐soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson‐Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme‐heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new‐to‐nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.