Inspection of heme protein structures in the protein data bank reveals four isomers of heme characterized by different relative orientations of the vinyl side chains; remarkably, all these have been reported in multiple protein structures. Density functional theory computations explain this as due to similar energy of the isomers but with a sizable (25 kJ mol⁻¹) barrier to interconversion arising from restricted rotation around the conjugated bonds. The four isomers, EE, EZ, ZE, and ZZ, were then investigated as 4-coordinate hemes, as 5-coordinate deoxyhemes, in 6-coordinate O₂-adducts of globins and as compound I intermediates typical of heme peroxidases. Substantial differences were observed in electronic properties relevant to heme function: notably, the spin state energy gap of O₂-heme adducts, important for fast reversible binding of O₂, depends on the isomer state, and O₂-binding enthalpies change by up to 16 kJ mol⁻¹; redox potentials change by up to 0.2 V depending on the isomer, and the doublet–quartet energy splitting of compound I, central to “two-state” reactivity, is affected by up to ∼15 kJ mol⁻¹. These effects are consistently seen with three distinct density functionals, i.e. the effects are not method-dependent. Thus, the nature of the isomer state is an important but overlooked feature of heme chemistry and function, and previous and future studies of hemes may be reconsidered in this new context.