NITROGEN AND PROTON ENDOR OF CYTOCHROME-D, HEMIN, AND METMYOGLOBIN IN FROZEN-SOLUTIONS

Orientation-selected electron nuclear double resonance (ENDOR) spectra have been obtained from iron-linked nitrogens of the d heme of the cytochrome d oxidase complex in frozen solution. Heme d is a high-spin chlorin moiety which exists in the cytochrome d complex located in the inner membrane of Escherichia coli. This complex is a terminal oxidase in the E. coli aerobic respiratory chain. Comparison of the spectra of cytochrome d with hemin and metmyoglobin strongly implies that the d heme does not contain an axial nitrogen ligand. By computer simulation of the ENDOR spectra for each magnetic field, the nitrogen hyperfine interaction matrices and quadrupole coupling tensors to the three heme groups were determined. For each of the three heme groups studied, the proton hyperfine coupling perpendicular to the heme plane was also measured. From average hyperfine coupling constants, the unpaired electron densities in the nitrogen 2s and 2p valence orbitals were calculated with standard ligand field techniques. The quadrupole constants are also related to electron populations in the nitrogen orbitals. The comparison between theoretical calculations and experimental data is shown.