OBSERVATION OF A NEW OXYGEN-ISOTOPE-SENSITIVE RAMAN BAND FOR OXYHEMOPROTEINS AND ITS IMPLICATIONS IN HEME POCKET STRUCTURES

A new oxygen-isotope-sensitive Raman band was found for oxyhemoglobin (HbO(2)) and oxycytochrome c oxidase (CcO.O-2) in the frequency region lower than the Fe-O-2 stretching mode (nu(Fe-O2)). This band was located at 425 cm(-1) for Hb(16)O(2) and shifted to 405 cm(-1) with Hb(18)O(2) and to similar to 423 and similar to 407 cm(-1) with Hb(16)O(18)O. Th, corresponding band appeared at 435 cm(-1) for CCO.O-16(2) and shifted to 415 cm(-1) with CCO.O-18(2), Accordingly, the band has been assigned to the Fe-O-O bending mode (delta(FeOO)) However, the corresponding band could not be identified for oxymyoglobin (MbO(2)). The Fe-On stretching mode (nu(Fe-O2)) was observed at 568, 567, 544, and 544 cm(-1) for Hb(16)O(2), Hb(16)O(18)O(2), Hb(18)O(16)O(2), Hb(18)O(2), respectively, and the corresponding modes were observed at 571, 569, 547, and 545 cm(-1) for MbO(2) and 571, 567, 548, and 544 cm(-1) for CcO.O-2. The nu(Fe-O2) bandwidths of HbO(2) and MbO(2) were alike and 1.5 times broader than that of CcO.O-2, suggesting that the Fe-O-O geometry is more nearly fixed in the latter. Despite the greatly different reactivities of bound O-2 in HbO(2) and CcO.O-2, their nu(Fe-O2) and delta(FeOO) frequencies and O-2-isotopic frequency shifts were alike, indicating similar Fe-O-O binding geometries. Normal coordinate calculations for an isolated three-atom molecule could reproduce the observed isotopic frequency shifts with the 115 degrees bond angle reported for MbO(2), but not with the 155 degrees angle reported for HbO(2).