Heme-Nitric oxide and/or OXygen binding (H-NOX) proteins are a family of recently discovered heme-based sensor proteins for diatomic molecules. Crystal structures of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) have greatly aided the study of the molecular basis of diatomic molecule sensing by H-NOX proteins. In our work, we employ X-ray Absorption Spectroscopy (XAS) to study the local electrostatic and structural properties of the heme-iron center in H-NOX proteins. XAS spectra of various ligands bound to H-NOX have been analyzed. The iron K-edges indicate effective charges on the iron center and report on the electronic environment of heme iron. The ligand field indicator ratio (LFIR), which is extracted from the X-ray Absorption Near-Edge Structure (XANES) analysis, provides an understanding of ligand field strength, spin state of the central iron, movement of the iron atom upon ligation, and ligand binding properties for each complex. In particular, the LFIRs demonstrate that the heme iron is dramatically displaced towards the distal pocket when ligand binds, which is expected to be the mechanistic link between ligand binding and subsequent changes in heme and protein conformation. Furthermore, bond lengths and bond angles of the H-NOX complexes were determined by analyzing the Extended X-ray Absorption Fine Structure (EXAFS) spectra, providing detailed structural information into the heme pocket. These results are extrapolated to gain insight into changes in heme and protein conformation during a signaling event. It is also discovered that Tt H-NOX can be used to selectively detect cyanide with a limit as low as 0.5 ÇªM and an upper detection range that is adjustable with protein concentration. Engineering of the cyanide binding pocket through site-directed mutagenesis improves the detection selectivity and enables the visual detection of cyanide with this sensing system. Thus we demonstrate the ability to fine-tune the affinity and selectivity of Tt H-NOX for cyanide, suggesting that Tt H-NOX can be readily tailored into a practical cyanide sensor. Here we present our efforts to describe the molecular details that dictate small molecule sensing by H-NOX proteins.