Vibrational circular dichroism measurements of ligand vibrations in haem and non-haem metalloenzymes

Abstract

We have measured the vibrational circular dichroism (VCD) spectra of the stretching vibrations of azide and cyanide ligated to the Fe³⁺ atoms of haemoglobin (Hb) and myoglobin (Mb). The antisymmetric azide-stretch of the low-spin haems have an anomalously large g-value of ca.–1 × 10^–3. In contrast, CN^– has a g-value of ca.+2.4 × 10^–3. We also show, for the first time, that a significant VCD occurs for the azide ligand antisymmetric stretches of non-haem proteins; we measure a g-value of ca.–1 × 10^–4 for azide bound to haemerythrin. We have examined the mechanism of the VCD phenomenon by: (1) reconstituting Mb with haems substituted such that they insert differently in the haem pocket; (2) replacing the Fe³⁺ with Mn³⁺; (3) examining proteins where replacements occur for E-7 His and E-11 Val distal amino acids close to the haem and (4) examining an Mb mutant where the proximal F-8 His is replaced by Gly, and where an imidazole ligand inserts into the resulting crevice and binds to the haem in a way similar to that of the proximal histidine in the native protein. The VCD anisotropy appears insensitive to the haem substituent replacements used in this study. Exchange of the E-7 distal His or the E-11 Val has a dramatic effect on the g-value. Exchange of the F-8 proximal His reverses the sign of the g-value for the azide complex, but not for the cyanide complex. The work to date indicates that VCD has the potential to become a sensitive technique for examining the structure of metalloenzymes. Work is needed to determine the mechanism giving rise to the large g-values and to correlate the VCD spectrum with the metalloenzyme structure at the active site.