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

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 C1 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.