Date of Award

Spring 1990

Project Type

Dissertation

Program or Major

Chemistry

Degree Name

Doctor of Philosophy

First Advisor

N Dennis Chasteen

Abstract

Information about the ligand environment of the iron binding sites in a cyanide adduct of transferrin was obtained by an analysis of powder-type ENDOR and ESEEM spectra. The low-spin cyanide adduct of transferrin is formed only in the C-terminal site of the protein and is characterized by a rhombic EPR spectrum. Earlier work demonstrated that three cyanide groups are necessary to form the adduct, but it wasn't clear whether these groups were coordinated directly to the metal or to cationic sites on the protein. The ENDOR spectra of the $\sp{13}$CN adduct showed one set of $\sp{13}$C ENDOR resonances which probably corresponds to only one or two of the CN groups, the other(s) being ENDOR silent. Simulations of the ENDOR line positions indicate a substantial isotropic coupling and smaller dipolar couplings. From an analysis of the orientation dependent dipolar term it is concluded that the carbon giving rise to the ENDOR signals lies along the g$\sb{\rm xx}$ axis of the g-tensor. The iron-carbon distance was calculated based on a point dipole model and a model in which the ground state metal-based d$\sb{\rm xy}$ orbital of the electron was considered explicitly. ESEEM studies with C$\sp{15}$N indicated that there are at least two equivalent CN groups coordinated to the iron center. The maximum hyperfine coupling for the nitrogen was observed near g$\sb{\rm xx}$ which is consistent with the ENDOR results which placed the carbon of the cyanide group on the g$\sb{\rm xx}$ axis. There is a substantial hyperfine contribution along g$\sb{\rm zz}$ which indicates that the CN bond is not aligned along the g$\sb{\rm xx}$ axis. The lack of resolved couplings in the spectrum along g$\sb{\rm yy}$ makes it impossible to determine the nitrogen position exactly. The ESEEM spectra of the C$\sp{14}$N adduct were complicated, presumably because the Zeeman and hyperfine terms did not cancel at the spectrometer frequencies used. The ESEEM and ENDOR spectra of the D$\sb2$O solvent exchanged adduct demonstrated that water does not coordinate directly to the iron. The ESEEM of the g$\sp\prime$ = 4.3 signal which arises from the high-spin N-terminal site was also studied and the observed ESEEM resonances were attributed to histidine nitrogen.

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