SEQUENTIAL-CHANGES OF THE FE-HISTIDINE BOND UPON LIGAND-BINDING TO HEMOGLOBIN - RESONANCE RAMAN-STUDY OF ALPHA,ALPHA-CROSS-LINKED CO-FE HYBRID HEMOGLOBINS

Resonance Raman spectra were observed for alpha,alpha-cross-linked Co- Fe hybrid Hbs. The Fe-histidine (F8) stretching (nu(Fe-His)), Fe-CO st retching (nu(Fe)-CO), Fe-C-O bending (delta(FeCO)), and O-O stretching (nu(OO)) frequencies were determined for mono-, di-, and tri-Fe tetra mers in a function of the number of bound ligands. The nu(Fe-His) band center was determined with the accuracy of +/-0.25 cm(-1), while unce rtainties of the nu(Fe-CO) and nu(OO) frequencies were +/-1 cm(-1). Th e nu(Fe-His) band of alpha,alpha-cross-linked Hb appeared at 214 and 2 20 cm(-1) for the equilibrium deoxy and CO-photodissociated transient forms (an average within 50 mu s following photolysis), respectively, in good agreement with those of Hb A. The nu(Fe-His) bands of mono- an d di-alpha(Fe) tetramers were alike and were composed of two bands irr espective of the number of bound ligands, and they were significantly lower than those of mono- and di-(deoxy)beta(Fe) subunits, which gave an intense symmetric band, indicating inequivalence between alpha and beta subunits. This feature was qualitatively retained while the frequ encies of two alpha(Fe) hemes and their relative intensities were slig htly changed in the binding process of four ligands. In contrast, the nu(Fe-His) frequency of the beta subunits changed gradually as the num ber of bound ligands increased. The nu(Fe-His) frequency of alpha(Fe) was lower by 4-6 cm(-1) in the alpha,alpha- than in the beta,beta-cros s-linked Co-Fe hybrid Hb, while those of beta(Fe) were alike. The nu(F e-CO) frequency was higher for the alpha(Fe) than beta(Fe) subunits by 2-4 cm(-1) for both mono- and di-Fe species, and changed a little wit h the number of bound ligands for the alpha subunit but not for the be ta subunit. The nu(OO) frequency of the oxyCo subunit was scarcely alt ered between the alpha and beta subunits and also with the number of b ound O-2 ligands. The present results about nu(Fe-Co) and nu(OO) bands were in agreement with the results previously obtained for the beta,b eta-cross-linked Co-Fe hybrid Hb, suggesting that structural changes u pon the increase in the number of the bound ligands are much larger in the proximal side than in the distal side. The behavior of the nu(Fe- His) band suggests that the proximal structure little changes in the a lpha subunit until binding of two ligands but that in the beta subunit starts to change from the first ligand.