CYP119, a bacterial thermophilic protein from the cytochrome P450 superfamily, has previously been observed in three different conformations with different inhibitors bound using X-ray crystallography. The significance of these states in solution and in the function of the enzyme is not well-known. Double electron-electron resonance (DEER) was used to measure distances and distance distributions between spin-labels for populated conformational states in solution. DEER spectroscopy and molecular dynamics for the ligand-free enzyme suggest that the G helix is in a slightly different conformation than seen previously by crystallography, with the F/G loop in a slightly open conformation. Inhibitor-bound samples showed that this conformation remains as the predominant form, but partial conversion is indicated to a more closed conformation of the F/G loop. However, when the enzyme binds to lauric acid, the proposed substrate, it induces the conversion to a state that is characterized by increased disorder. We propose that similar to recent results with soluble CYP3A4, binding of the inhibitor to CYP119 is accompanied by only small changes in the enzyme structure, but substrate binding results in greater heterogeneity in the structure of the F/G loop region.