Investigations of cyanide as an infrared probe of hemeprotein ligand binding sites.

Abstract

The measurement of infrared spectra for cyanide liganded to hemeproteins and hemins has been investigated. The hemeproteins included human methemoglobin A, lamprey methemoglobin, metchlorocruorin, horse metmyoglobin, and horseradish peroxidase. The hemins were dicyanide and monopyridine monocyanide species of deuteroporphyrin IX iron(III) and its 2,4-divinyl(proto) and 2,4-diacetyl derivatives. C-N stretch bands of low intensity detected near 2100 cm-1 exhibit changes in frequency, width, intensity, and isotope shift with changes in cyanide compound structure. Infrared band parameters are particularly sensitive to a change in oxidation state (Fe2+ versus Fe3+) and are affected to a lesser extent by changes in porphyrin ring substituent, ligand trans to the cyanide, and protein structure. Evidence of multiple conformers (i.e. multiple C-N stretch bands) was found for several hemeproteins. The cyanide infrared spectra provide direct evidence for cyanide binding as a metal cyanide (Fe--C identical to N) and against HCN being the ligand in nitrile-like bonding (Fe--N identical to C--H) in all the hemeprotein and hemin cyanides studied. With the reduced horseradish peroxidase cyanide, differences between infrared spectra for D2O and H2O solutions can result from hydrogen bonding between a protein amino acid residue and the distal atom of the cyanide (Fe--C identical to N...H+--R). The binding of cyanide to reduced iron (Fe2+) of a hemeprotein was only observed in the case of the reduced peroxidase. These findings demonstrate that cyanide infrared spectra can not only determine when cyanide is bound to a metalloprotein but can also provide information on how the cyanide is bonded to metal and on characteristics of the ligand binding site.