Probing Structural and Electronic Parameters in Randomly Oriented Metalloproteins by Orientation-Selective ENDOR Spectroscopy

Abstract

Electron nuclear double resonance (ENDOR) spectroscopy in its continuous wave (CW) and pulsed operational modes is now widely used to characterize the functional, structural, and electronic properties particularly of paramagnetic centers found in metalloproteins. Its essential advantage is based on the intrinsic high resolution, which permits the analysis of small interactions of the metal nucleus or of nuclei in the vicinity of the metal center with the electron spin not observable by standard EPR techniques. Because most of the protein samples are available only as frozen solutions, the essential concept introduced for the analysis of such “powder”-type ENDOR spectra is a method for calculation of “orientation-selected” ENDOR spectra. In this approach the EPR resonance condition is solved for a set of g-, hyperfine-tensors of the paramagnetic center(s), yielding the orientational distribution of the subset of molecules contributing to the ENDOR spectrum recorded at a certain magnetic field value. For these orientations the ENDOR transitions can be calculated and the ENDOR powder spectrum composed. Typically, the resonance lines arise from cumulations (turning points) of the selected orientations in defined frequency ranges. By recording several ENDOR spectra across an EPR spectrum, the tensors of the interactions, not resolvable in EPR, are probed.