Infrared crystallographic investigation of T-state hemoglobin

Polarized IR measurements on single crystals of human hemoglobin, grown under low salt conditions that stabilize the T quaternary structure, allow spectral features to be associated with individual sites within the molecule. Differences between the a- and c-polarized IR spectra in the sulfhydryl stretching region distinguish the contributions of individual Cys residues to the S-H band and lead to an evaluation of possible H-bonding partners for Cys beta-112. Successful modelling of both crystal and solution S-H spectra with component bands having identical frequencies and bandwidths, supports the use of the X-ray structure as a model for the low affinity T-state in solution. Polarization analysis of a crystal partially saturated with CO reveals comparable occupancy of the alpha- and beta-hemes. In the T-state crystal, the C-O bands are broader and lower in frequency than in the R-state solution, and 20% of the CO-ligated beta-subunits adopt an alternate conformation with a 1967 cm(-1) C-O frequency. The latter observation reflects an energetically significant disruption of the distal heme pocket upon CO binding to beta-hemes in the low affinity T-state.