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 from a proton of the functionally important Phe14 ring, 3.3 Å away from the heme iron, was detected. ENDOR of the nitrogen ligand hyperfine structure is a direct probe into the σ-antibonding (dz2) and (dx2-y2) orbitals whose energies alter the relative stability and admixture of sextet and quartet states and whose electronic details were thus elucidated. ENDOR frequencies showed for cytochrome c‘ larger hyperfine couplings to the histidine nitrogen and smaller hyperfine couplings to the heme nitrogens than for high-spin ferric hemes. Both of these findings followed from the mixed-spin ground state, which has less (dx2-y2) character than have fully high-spin ferric heme systems.
JACS, 2005, V.127, No.26, pp.9485-9494