Die Fe2+-His(F8)-Resonanz-Ramanbande der Nachweis fu?r taxonomische Konformationssubzusta?nde in der proximalen Bindung


Abstract (Summary)
The Iron-Histidine Resonance Raman Band of Heme Proteins: Implications for Taxonomic Conformational Substates of the Proximal Linkage The band shape of the Raman line attributed to the Fe2+-N?(His-F8) stretching mode in heme proteins contains significant information of the Fe-His proximal linkage. In the case of myoglobin Raman lines at the low and the high frequency wing of this profile obscure its true band shape. Measurements with different pH-values, different solvents and different polarizations showed that these lines are not caused by the Fe-His-linkage. In order to separate these accompanying lines from the Fe-His-band the isotopic shift has been used by substituting the natural iron-isotope 56Fe of the heme with the 54Fe-isotope. This enables us to isolate the true line shape. The ?(Fe-His)-band of sperm whale myoglobin dissolved in 66%|V ol/V ol glycerol/water solution is measured for nine temperatures from 10K to 270K. The isolated profile shows a complex temperature-dependence, with a shoulder on its high-frequency wing, which becomes more prominent with increasing temperature. Detailed analysis reveals that the band is composed of five distinct lines attributable to taxonomic conformational substates of the Fe-His linkage. The temperature dependence of the band is caused by intensity variations of these five different sublines. This result of the analysis is confirmed by measurements on sperm whale and horse heart myoglobin in glycerol/water solution and in water. Additionally the measurements were performed also on the isolated subunits of hemoglobin, the ?- and the ?-chains. The most important advantage of those is the lack of the accompanying lines on the high and low frequency wings. The profiles of their ?(Fe-His)-bands show several shoulders at low temperatures. The analysis of these profiles point out that the ?(Fe-His)-band of the ?-chains consist of four and that of the ?-chains of five sublines, which are also attributed to conformational substates of the heme-protein linkage in these proteins. All measurements on the different proteins were performed with the excitation wavelength of 441.6nm and are repeated with the wavelength at ?=413.1nm. In all cases, the band shape at the excitation wavelength of 413.1nm differs from that at the wavelength of 441.6nm. This was interpreted by different resonant raman enhancement of the various sublines, which are attributed to the different substates in the Fe-His linkage. The temperature dependence of the sublines was explained in terms of a thermodynamic model, in which the intensity of a certain subline is proportional to the occupation of the corresponding substate. The intensity ratio of such two successive sublines exhibits a van’t Hoff behavior between 270K and 180K bending over in region from 180K to 100K and remaining constant at temperatures below than 100K. This behavior can be fitted by a modified van’t Hoff expression, which accounts for the freezing into a non-equilibrium distribution at temperatures below a distinct transition temperature Tf . The fits evaluate a transition temperature of Tf =155±15K and a relatively broad transition region of ?T?80K for myoglobin and the ?-chains. The enthalpic differences between conformational substates yield values between 0.7kJ/mol and 1.5kJ/mol for myoglobin, whereas the ?-chains show enthalpies between 1.3kJ/mol and 3.6kJ/mol. The ?-chains exhibit a lower transition temperature of Tf ?130K and enthalpies between 0.5 and 1.7kJ/mol, similar to those of myoglobin. Our data show, that the ?(Fe-His) vibration is governed by at least one coordinate x which determines its frequency. This can be the tilt angle ?, or the displacement ? of the central iron or a combination of both. The sublines are therefore interpreted as resulting from different conformational substates of the Fe2+-N?(His-F8) complex which differ in terms of x and consequently also in their energies. The success of the model of conformational substates in the proximal linkage in all examined he- me proteins excludes the interpretation according to alternative models, which try to explain the ?(Fe-His) band shape by either an anharmonic coupling of the Fe-His vibration to a low-frequency heme doming mode (Biophys. J., 77, 2764, Bitler and Stavrov, 1999) or by a Gaussian distribution of conformational substates of the iron out-of-plane displacements (Phys. Rev. Let., 57, 1267, ? Srajer et al., 1986). In conclusion the results of this work prove the existence of taxonomic substates in the Fe2+-N?(His-F8) linkage in heme-proteins. Inhaltsverzeichnis 1 Einleitung 1 2 Grundlagen 3 2.1 Proteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2 Funktion und Struktur des Myoglobins und des Hämoglobins
Bibliographical Information:


School:Hochschule Bremen

School Location:Germany

Source Type:Master's Thesis



Date of Publication:

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