X-ray absorption spectroscopic studies of a transient intermediate in the reaction of cyanide metmyoglobin with dithionite by using rapid freezing

Abstract

The reaction of cyanide metmyoglobin (Mb +CN −) with dithionite produces a transient intermediate, supposed to be cyanide-ligated ferrous myoglobin. The Fe K-edge X-ray absorption spectrum of the intermediate has been measured by using rapid freezing and compared with those of Mb +CN − and deoxymyoglobin (deoxyMb). The shapes of the XANES (X-ray Absorption Near Edge Structure) spectra of Mb +CN − and the intermediate are very similar, including the intensity ratios of the peak C 1 to D. This indicates that CN − remains bound with a linear FeCN configuration in the intermediate. The absorption edge of the intermediate is shifted to 1.2 eV lower energy than that of Mb +CN −, reflecting a valence change in the heme iron. The EXAFS (Extended X-ray Absorption Fine Structure) spectrum of the intermediate closely resembles that of Mb +CN − but significantly differs from that of deoxyMb. Analysis shows that the average iron-nearest neighbor atom distance is 1.99 ± 0.01 A ̊ for both Mb +CN − and the intermediate and 2.05 ± 0.01 A ̊ for deoxyMb. These results imply that the local structure around the heme iron of Mb +CN − does not change upon reduction until the cyanide ligand is released.