Iron-carbon bond lengths in carbonmonoxy and cyanomet complexes of the monomeric hemoglobin III from Chironomus thummi thummi: a critical comparison between resonance Raman and x-ray diffraction studies

Soret-excited resonance Raman spectroscopy yields direct information regarding the iron-carbon bonding interactions in the cyanomet and carbonmonoxy complexes of hemoglobin III from Chironomus thummi thummi (CTT III) in solution. By isotope exchange in cyanide (13CN-, C15N-, and13C15N-) and carbon monoxide (13CO, C18O, and13C18O), we have assigned the Fe(III)--CN-stretching at 453 cm-1, the Fe(III)--C--N-bending at 412 cm-1, the Fe(II)--CO stretching at 500 cm-1, the Fe(II)--C--O bending at 574 cm-1, and the C--O stretching at 1960 cm-1. The resonance Raman data, in conjunction with those obtained from heme model complexes with well-known Fe--C bond distances, strongly suggest that the Fe(III)--CN-bond (≈ 1.91 angstrom) is longer (hence weaker) than the Fe(II)--CO bond (≈ 1.80 angstrom). This result disagrees with those of x-ray crystallographic studies [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309-338] in which the Fe--C bond lengths were reported as 2.2 angstrom in cyanomet and 2.4 angstrom in carbonmonoxy CTT III. Based on Badger's rule and normal mode calculations, the x-ray data would lead to the prediction of 279 cm-1for the Fe(II)--CO stretching frequency in CTT III angstrom CO, which was not observed. On the other hand, we estimate the Fe--CO bond as ≈ 1.82 angstrom, which is very similar to the 1.80- angstrom value in human Hb· CO crystals. Furthermore, we have used isotope shift data to estimate the Fe--C--O angle as 169 ± 5 degrees, somewhat larger than the 161 degrees value found by Steigemann and Weber. We therefore conclude that there must be errors in the x-ray crystallographic refinement for the ligand geometry in carbonmonoxy and cyanomet CTT III.