ENDOR Investigation of the Liganding Environment of Mixed-Spin Ferric Cytochrome c

The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) heme center in cytochrome c‘ was investigated by electron nuclear double resonance (ENDOR), a technique not previously applied to this mixed-spin system. Cytochrome c‘ was obtained from overexpressing variants of Rhodobacter sphaeroides 2.4.3. ENDOR for this study was done at the g|| = 2.00 extremum where single-crystal-like, well-resolved spectra prevail. The heme meso protons of cytochrome c‘ showed a contact interaction that implied spin delocalization arising from the heme (dz2) orbital enhanced by iron out-of-planarity. An exchangeable proton ENDOR feature appeared from the proximal His123 Nδ hydrogen. This Nδ hydrogen, which crystallographically has no hydrogen-bonding partner and thus belongs to a neutral imidazole, showed a larger hyperfine coupling than the corresponding hydrogen-bonded Nδ proton from metmyoglobin. The unique residue Phe14 occludes binding of a sixth ligand in cytochrome c‘, and ENDOR fr...