The carbon monoxide derivative of human hemoglobin carrying the double mutation LeuB10→Tyr and HisE7→Gln on α and β chains probed by infrared spectroscopy

The fine structural properties of the distal heme pocket have been probed by infrared spectroscopy of ferrous carbon monoxy human hemoglobin mutants carrying the mutations LeuB10→Tyr and HisE7→Gln on the α, β, and both chains, respectively. The stretching frequency of iron-bound carbon monoxide occurs as a single broad band around 1943 cm−1 in both the α and the β mutated chains. Such a frequency value indicates that no direct hydrogen bonding exists between the bound CO molecule and the TyrB10 phenolic oxygen, at variance with other naturally occurring TyrB10, GlnE7 nonvertebrate hemoglobins. The rates of carbon monoxide release have been determined for the first time by a Fourier transform infrared spectroscopy stopped-flow technique that allowed us to single out the heterogeneity in the kinetics of CO release in the α and β chains for the mutated proteins and for native HbA. The rates of CO release are 15- to 20-fold faster for the mutated α or β chains with respect to the native ones consistent with the lack of distal stabilization on the iron-bound CO molecule. The present results demonstrate that residues in key topological positions (namely E7 and B10) for the distal steric control of the iron-bound ligand are not interchangeable among hemoglobins from different species.