ENDOR investigation of the liganding environment of mixed-spin ferric cytochrome c'

摘要

The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quarte) 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(parallel to) = 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 (d(z2)) orbital enhanced by iron out-o planarity. An exchangeable proton ENDOR feature appeared from the proximal His123 N delta hydrogen. This N delta 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 N delta 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, similar to 3.3 angstrom away from the heme iron, was detected ENDOR of the nitrogen ligand hyperfine structure is a direct probe into the sigma-antibonding (d(z2)) and (d(x2-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 (d(x2-y2)) character than have fully high-spin ferric heme systems.