The Reactivity with CO of AHb1 and AHb2 from Arabidopsis thaliana is Controlled by the Distal HisE7 and Internal Hydrophobic Cavities

Using steady-state and time-resolved spectroscopic methods, we show that Fe2+ AHb1 contains a mixture of penta- and hexacoordinated heme, while Fe2+ AHb2 is fully hexacoordinated. In the CO complexes, polar interactions and H-bonds with the ligand are stronger for AHb1 than for AHb2. The ligand binding kinetics are substantially different, reflecting the distribution between the penta- and hexacoordinated species, and indicate that protein dynamics and ligand migration pathways are very specific for each of the two proteins. Overall, these kinetic and structural data are consistent with the putative NO-dioxygenase activity previously attributed to AHb1, whereas the role of AHb2 remains elusive