Determination of Fe-CO geometry in the subunits of carbonmonoxy hemoglobin M Boston using femtosecond infrared spectroscopy.

We have undertaken ultrafast infrared (IR) spectroscopic studies in order to elucidate the geometry of bound CO in the alpha and beta subunits of hemoglobin (Hb) M Boston 13CO. Hb M Boston is a mutant human Hb in which the distal histidine in the alpha subunits is replaced by a tyrosine. The IR absorptions of bound 13CO fall at 1925 cm-1 for the alpha subunits and 1907 cm-1 for the beta subunits. Despite a difference of nearly 20 cm-1 in these peaks, the measured anisotropies of the bound 13CO depletions following 30% photolysis are nearly identical, with values of -0.142 +/- 0.002 obtained for the alpha subunits and -0.140 +/- 0.003 obtained for the beta subunits. These translate to values of 20 degrees +/- 1 degree and 21 degrees +/- 1 degree for the values of the average angles between the CO bond and the normal to the heme planes in the alpha and beta subunits, respectively. Our present results and the work of previous investigators [Nagai, M., Yoneyama, Y., & Kitagawa, T. (1991) Biochemistry 30, 6495-6503] suggest that a change in the polar interactions of the bound CO with the heme pocket environment upon substitution of tyrosine for the distal histidine and a less bent structure for the Fe-C-O unit in the alpha subunits are responsible for the difference in the bound CO absorption frequencies in the alpha and beta subunits. A spectrum of the depletion of the bound 13CO peaks following photolysis indicates that both subunits photodissociate CO with the same quantum yield and neither subunit exhibits significant recombination within 1 ns.