Sampling Field Heterogeneity at the Heme of c-Type Cytochromes by Spectral Hole Burning Spectroscopy and Electrostatic Calculations

Abstract

We report on a comparative investigation of the heme pocket fields of two Zn-substituted c-type cytochromes-namely yeast and horse heart cytochromes c-using a combination of hole burning Stark spectroscopy and electrostatic calculations. The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochromes. In the case of horse heart Zn-cytochrome c, two distinguishable electronic origins with different electrostatic properties are observed. The yeast species, on the other hand, displays a single electronic origin. Electrostatic calculations and graphics modeling using the linearized finite-difference Poisson-Boltzmann equation performed at selected time intervals on nanosecond-molecular dynamics trajectories show that the hemes of the respective cytochromes sample different potentials as they explore conformational space. The electrostatic potentials generated by the protein matrix at the heme show different patterns in both cytochromes, and we suggest that the cytochromes differ by the number of "electrostatic substates" that they can sample, thus accounting for the different spectral populations observed in the two cytochromes.

Spectroscopy of proteins at low temperature. Part I: Experiments with molecular ensembles

“…The Stark-spectra show rather convincingly that it is the red conformation which does not seem to be populated in cytochrome c from yeast. These findings are strongly supported by MD-simulations and electrostatic calculations which demonstrate that the conformational phase space sampled by the cytochrome c from horse heart is significantly larger than the respective one sampled by cytochrome c from yeast [70].…”

The importance of vibronic perturbations in ferrocytochrome c spectra: A reevaluation of spectral properties based on low-temperature optical absorption, resonance Raman, and molecular-dynamics simulations

“…They showed that, for the same quadrupole coupling, yc exhibits a substantially lower splitting than hhc, owing to the fact that the difference ‫ץ‬E x / ‫ץ‬x − ‫ץ‬E y / ‫ץ‬E y is much smaller in the former, as shown earlier by Laberge et al 5 ent of the electric field at the heme. This is in line with our earlier prediction that a nonuniform rather than a uniform field causes the splitting.…”

“…Additionally, the internal electric field created by ionizable and polar side chains causes a perturbation of the heme's potential energy. [2][3][4][5][6][7] Numerous studies have been performed to explore the influence of the proximal and distal ligands on the oxidation and spin state of the central iron atom. The proximal ligand ͑histidine in most cases͒ was found to exert a particularly strong influence on the heme iron as well as on the symmetry of the heme macrocycle.…”

Cytochrome c: A Multifunctional Protein Combining Conformational Rigidity with Flexibility

“…Fluorescence hole burning experiments on heme proteins with Zn-substituted heme groups revealed electric field strengths in the range of 10 7 /cm. Theoretical calculations based on the solution of Laplace equations yielded values in the same order of magnitude [36]. Manas et al argued on theoretical grounds that this internal electric field can cause a splitting of both the -and -band, if the field vector does not exactly bisect the two transition dipole moments of the twofold degenerate electronic transitions [35].…”

“…In spite of a rather asymmetric heme environment and strong heme-protein interactions via the two axial ligands [10,30,31], the thioether bridges [32], the internal electric field at the heme [33][34][35][36][37][38], and to a lesser extent substituent-hydrogen bonding [25,26,[39][40][41] and multiple van der Waals contacts [42], spectroscopists have for a long period of time considered the heme as exhibiting an ideal D 4h -symmetry. This was (and in part still is) particularly true for the interpretation of optical absorption and resonance Raman data [43][44][45][46][47][48][49].…”