DETERMINATION OF FE-CO GEOMETRY IN THE SUBUNITS OF CARBONMONOXYHEMOGLOBIN M BOSTON USING FEMTOSECOND INFRARED-SPECTROSCOPY

We have undertaken ultrafast infrared (IR) spectroscopic studies in or der to elucidate the geometry of bound CO in the alpha and beta subuni ts of hemoglobin (Hb) M Boston (CO)-C-13. Hb M Boston is a mutant huma n Hb in which the distal histidine in the a subunits is replaced by a tyrosine. The IR absorptions of bound (CO)-C-13 fall at 1925 cm-1 for the alpha subunits and 1907 cm-1 for the beta subunits. Despite a diff erence of nearly 20 cm-1 in these peaks, the measured anisotropies of the bound (CO)-C-13 depletions following 30% photolysis are nearly ide ntical, with values of -O.142 +/- 0.002 obtained for the alpha subunit s and -0.140 +/- 0.003 obtained for the beta subunits. These translate to values of 20-degrees +/- 1-degrees and 21-degrees +/- 1-degrees fo r 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 p resent results and the work of previous investigators [Nagai, M., Yone yama, Y., & Kitagawa, T. (1991) Biochemistry 30, 6495-6503] suggest th at a change in the polar interactions of the bound CO with the heme po cket environment upon substitution of tyrosine for the distal histidin e 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 frequenc ies in the alpha and beta subunits. A spectrum of the depletion of the bound (CO)-C-13 peaks following photolysis indicates that both subuni ts photodissociate CO with the same quantum yield and neither subunit exhibits significant recombination within 1 ns.