JBIC (1997) 2 : 516–520 Q SBIC 1997 COMMENTARY Thomas G. Spiro 7 Pawel M. Kozlowski Will the real FeCO please stand up? Received, accepted: 23 May 1997 T. G. Spiro (Y) 7 P. M. Kozlowski Department of Chemistry, Princeton University, Princeton, NJ 08544, USA Fax: (609)258-0348; e-mail: spiro@princeton.edu Abstract The paradigm that nature protects us from CO poisoning by forcing the bound CO to bend over in heme proteins, thereby reducing its binding affinity, is now in textbooks, but is nevertheless problematic. Re- sults from vibrational spectroscopy give no evidence for bent CO, although X-ray crystallography continues to indicate appreciable distortions in myoglobin. Howev- er, the energetic significance of the discrepancy is doubtful, since new Density Functional Theory calcula- tions indicate that much less energy is required to dis- tort the CO than had been thought, perhaps 2 kcal/mol or less. Binding studies on site-directed mutants of myoglobin suggest that steric hindrance by the distal histidine is worth ca. 1 kcal/mol. While the distal histid- ine does account for the discrimination by Mb against CO and in favor of O 2 , most of the effect is due to its H-bond with bound O 2 . Key words Myoglobin-CO 7 Bond distortion 7 Bond energy 7 CO poisoning 7 Hydrogen bond 7 Infrared 7 Raman Introduction Few matters have exercised bioinorganic chemists more than the issue of the CO-binding geometry in myoglo- bin (Mb) and other heme proteins. Despite the well- known propensity of CO to bind transition metal atoms in a linear fashion, crystal structure determinations of MbCO, from both neutron [1–4] and X-ray [5] diffrac- tion, found large deviations from linearity, F120–1407, presumably because of steric hindrance from the distal histidine residue, whose imidazole sidechain lies direct- ly over the heme. Twenty years ago, Collman et al. [6] proposed that steric hindrance serves to discriminate against CO and in favor of O 2 since the FeO 2 unit is naturally bent, and that we are thereby saved from as- phyxiation by our endogenous CO. This idea has made its way into textbooks [7], but it remains controversial. We are still uncertain of the energy value of the steric hindrance to CO binding, and we are still arguing over just how far the FeCO unit is distorted. The current situation can be summarized as follows: Site-directed mutagenesis [8] suggests that the distal histidine does hinder the binding of CO, but the effect is worth less than 1.6 kcal/mol. More important in dis- criminating against CO is the H-bond formed between the distal histidine and the bound O 2 , which is worth at least 2.3 kcal/mol. With respect to the structure of bound CO, different techniques give different answers. Several lines of evidence from vibrational spectroscopy argue for upright FeCO [6–10], while crystallography [1–4, 5, 14, 15] continues to show distortion. However, the direction and extent of distortion varies from one structure to another, and there seems to be no correla- tion with size of the distal group among site-mutants [14]. Finally, recent results from Density Functional Theory (DFT) [16] (P. M. Kozlowski and T.G. Spiro, unpublished) indicate that the distortion potential is softer than had been thought, and that quite modest protein forces could move the CO off-axis, when both tilting and bending coordinates are considered. Vibrational assignments and frequencies Three fundamental vibrational modes of the FeCO unit have been identified in heme protein resonance Raman (RR) spectra [15]: C-O stretching at ca. 1950 cm –1 , Fe- CO stretching at ca. 500 cm –1 , and Fe-C-O bending at ca. 575 cm –1 . The C-O stretch is also conveniently stud- ied by IR spectroscopy, but the n Fe-CO and d Fe-C-O modes are obscured by IR absorptions of the protein. The d Fe-C-O assignment has been controversial because this mode is of E symmetry in the idealized C 4v point