Sampling field heterogeneity at the heme of c-type cytochromes by spectralhole burning spectroscopy and electrostatic calculations

We report on a comparative investigation of the heme pocket fields of two Z n-substituted c-type cytochromes - namely yeast and horse heart cytochromes c - using a combination of hole burning Stark spectroscopy and electrostat ic calculations. The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochro mes. In the case of horse heart Zn-cytochrome c, two distinguishable electr onic origins with different electrostatic properties are observed. The yeas t species, on the other hand, displays a single electronic origin. Electros tatic calculations and graphics modeling using the linearized finite-differ ence Poisson-Boltzmann equation performed at selected time intervals on nan osecond-molecular dynamics trajectories show that the hemes of the respecti ve cytochromes sample different potentials as they explore conformational s pace. The electrostatic potentials generated by the protein matrix at the h eme show different patterns in both cytochromes, and we suggest that the cy tochromes differ by the number of "electrostatic substates" that they can s ample, thus accounting for the different spectral populations observed in t he two cytochromes.