Engineering of the heme pocket of an H-NOX domain for direct cyanide detection and quantification.
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.
Dai
Zhou
Z
Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
Boon
Elizabeth M
EM
D001665:Binding Sites; D003486:Cyanides / Q000032:analysis*; D006418:Heme / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D011487:Protein Conformation; D015202:Protein Engineering; D046968:Thermoanaerobacter / Q000737:chemistry*
D003486:Cyanides; D009569:Nitric Oxide; D006418:Heme; D010100:Oxygen