Engineering of the Heme Pocket of an H-NOX Domain for Direct Cyanide Detection and Quantification

Abstract

A new cyanide sensing system, the Heme-Nitric oxide and/or OXygen binding domain (H-NOX domain) from Thermoanaerobacter tengcongensis (Tt H-NOX), has been investigated. With straightforward absorbance-based detection, we have achieved a cyanide detection limit of 0.5 microM (approximately 10 ppb) with an upper detection range that is adjustable with protein concentration. We find a linear correlation of multiple spectroscopic features with cyanide concentration. These spectroscopic features include the Soret band maximum and absorbance changes in both the Soret and alpha/beta band regions of the spectrum. Multiple probes for cyanide detection makes sensing with Tt H-NOX unique compared to other cyanide sensing methods. Furthermore, using site-directed mutagenesis, we have rationally engineered the heme pocket of Tt H-NOX to improve its cyanide sensing properties. Using a mutant that alters the heme structure of Tt H-NOX (P115A) we were able to introduce colorimetric detection of cyanide. Through substituting phenylalanine 78 with a smaller (valine, F78V) or a larger residue (tyrosine, F78Y), we demonstrate a correlation with distal pocket steric crowding and affinity for cyanide. In particular, F78V Tt H-NOX shows a significant increase in CN(-) binding affinity and selectivity. Thus, we demonstrate the ability to fine-tune the affinity and specificity of Tt H-NOX for cyanide, suggesting that Tt H-NOX can be readily tailored into a practical cyanide sensor.

Unusual Heme-Binding PAS Domain from YybT Family Proteins

“…5B). Although cyanide binding often is observed only for ferric heme proteins (7,21), the addition of KCN to ferrous BsYybT 84-659 also resulted in small shifts in the Soret band (from 423 to 426 nm) and the ␤ and ␣ bands (from 557 and 528 nm to 561 and 529 nm, respectively) (Fig. 5B), suggesting that ferrous heme iron also forms a complex with CN Ϫ .…”

Mechanisms and Efficiency of the Simultaneous Removal of Metals and Cyanides by Using Ferrate(VI): Crucial Roles of Nanocrystalline Iron(III) Oxyhydroxides and Metal Carbonates

“…[6] New techniques have therefore been developed to detect low levels of cyanide and to detoxify cyanide in water. [7][8][9] Cyanide in solution exists in three forms: the free form, for example, HCN, CN À ; as a weak-acid dissociable form (WAD), for example, complexes of Cu, Zn, Cd, and Ni; and as strong-acid dissociable (SAD) cyanides, for example, complexes of Fe, Co, and Ag.…”

Conformational control of cofactors in nature – the influence of protein-induced macrocycle distortion on the biological function of tetrapyrroles

“…244,245 and in particular, for the highly non-planar conformation imposed by the protein on its single heme-cofactor. 246,247,248,249,250,251,252 This is one of the most distorted heme-cofactors to be observed in natural systems, 247 and this feature has been linked to its uncommonly high midpoint potential 248 and is likely crucial to its biological function. H-NOX proteins are proving to be particularly fruitful model systems that are being well explored.…”