An infrared study of carbon monoxide complexes of hemocyanins. Evidence for the structure of the co-binding site from vibrational analysis.

A vibrational analysis was carried out showing that the infrared experimental data of 13C and 18O carbon monoxide complexes of hemocyanin of Fager and Alben (Biochemistry 11 (1972) 4786) are consistent with a coordination of the carbon atom of CO to one of the two copper ions in the active site. This conclusion contradicts the original interpretation of Fager and Alben in which oxygen-coordination to copper was suggested. This vibrational analysis can also be applied to the study of Alben and Caughey (Biochemistry 7 (1968) 175) with 13C and 18O carbonyl hemoglobin, in which oxygen-coordination to iron was suggested. Carbonyl hemocyanins from several sources have also been studied by infrared spectroscopy. The single stretching vibration of CO bound to arthropodal (Cancer magister) hernocyanin (nu(co)) is at 2042.5 cm(-1), while nu(CO) for gastropod (Helix pomatia of the phylum Mollusca) alpha and beta hemocyanin is at 2064.5 cm(-1)and 2062.5 cm(-1), respectively. The intensities of the CO stretching bands were all around 1.5 X 10(4) M(-1) cm(-2). Calculations show that with the present attainable accuracy it is impossible to detect hydrogen bonding of exchangeable protons to small molecules bound to proteins (for example CO), by comparing its stretching frequencies in H2O and D2O buffers.