Stark effect experiments in cytochrome c-type proteins: structural hierarchies

Abstract

We performed hole-burning Stark effect experiments on cytochrome c in which the iron of the herne was either removed or replaced by Zn. According to the experiments, the free-base compound has an effective inversion center, even in the protein. The Zn compound, on the other hand, shows quite peculiar features: in the low-frequency range of the inhomogeneous band, it definitely has a dipole moment, as indicated by a splitting of the hole in the external field. However, in the maximum of the inhomogeneous band, a severe charge redistribution occurs, as the experiments show. In addition to the Stark experiments, we performed calculations of the electrostatic fields at the pyrrole rings and at the metal site of the heme group. We interpret our findings with a model based on structural hierarchies: the protein can exist in a few subconformations, which can be distinguished through the structure of the heme pocket. The different pocket structures support different structures of the chromophore, which, in turn, can be distinguished through their behavior in an external field. These distinct structures, in turn, correspond to a rather broad distribution of protein structures, which leave, however, the pocket structure largely unchanged. These structures show up in inhomogeneous broadening.

Using spectroscopic tools to probe porphyrin deformation and porphyrin-protein interactions

“…Spectral hole burning has emerged as a powerful method to probe the internal electric field at the active site of heme proteins [55,57]. However, its application requires the substitution of open shell (e.g.…”

Recombinant Cytochrome rC557 Obtained from Escherichia coli Cells Expressing a TruncatedThermus thermophilus cycA Gene

“…The energy and shape of the Q 00 band contains information about the structure and environment of the heme. This band is actually composed of two normally degenerate xy polarized transitions, which occur at different energies only when perturbations on the heme significantly lower its normal D 2h symmetry (43,44). The average energy of the Q 00 transitions depends on the nature of the heme substituents, to a lesser extent on the axial ligands of the iron, and to a much lesser extent on the dielectric environment.…”

Integrity of thermus thermophilus cytochrome c552 Synthesized by escherichia coli cells expressing the host‐specific cytochrome c maturation genes, ccmABCDEFGH: Biochemical, spectral, and structural characterization of the recombinant protein

“…Even though the absorption spectra are highly similar, it is important that the second derivatives of the reduced spectra also overlap particularly well across the Q 00 band. According to the ideas set forth by Vanderkooi and LaBerge~Kohler et al, 1996;Reddy et al, 1996! andShelnutt et al~Jentzen et al, 1998!, Q 00 splitting arises from the presence of an asymmetric electrostatic field at the heme and0or a combination of ruffling and saddling of the heme structure.…”