The cytochrome P450 superfamily of heme metalloenzymes catalyse important chemical reactions across nature. Understanding the substrate binding and the inhibition of these enzymes is critical for the development of drug molecules; for example, these are metabolised by mammalian forms of these enzymes or can target the P450s of pathogenic microbial species. The changes in the optical spectrum of P450s on addition of substrates (Type I) and inhibitors (Type II), are used to determine how molecules bind and assess the mechanism of inhibition. The current consensus view is that UV-Vis spectroscopy is not able to reliably differentiate between inhibitors which coordinate directly via a nitrogen donor atom from those whose nitrogen binds through a bridging water ligand. Therefore more complex methods have been used to distinguish these modes of substrate binding. Here we demonstrate that addition of related inhibitor molecules does result in distinct Type II UV-vis spectral responses. X-ray crystallography of the different inhibitor bound forms revealed that these differences arose when the nitrogen on the inhibitor bound directly to the heme iron or via a bridging iron-bound water ligand. Continuous wave EPR, HYSCORE and ENDOR data in frozen-solution revealed that the substrates bound in the active site of CYP199A4 in a form that is in agreement with the crystal structures and UV-vis spectra. The reduction potentials of the CYP199A4-substrate combinations provided further insight into the observed biochemical data and mechanism of inhibition. Overall, this study provides evidence that different binding modes result in distinguishable UV-vis spectroscopic responses and furthers our understanding of inhibitor binding in this important family of metalloenzymes. The explanation for previous work failing to distinguish between these modes is most likely due to complexity in substrate binding with multiple conformations being present.