The influence of protein environment on the low temperature electronic spectroscopy of Zn-substituted cytochrome c

Low-temperature UV-vis absorption and Stark-effect hole-burning spectra of Zn substituted cytochrome c are studied experimentally and theoretically us ing quantum mechanical and Poisson-Boltzmann electrostatics models. Both th e Q and Soret bands show resolved splitting at temperatures below similar t o 180 K. The trend observed in the splittings when comparing cytochromes fr om different species is found to be the same as that observed for the Q(0,0 ) band of ferrous cytochrome c. The relative magnitudes of the Q and Soret splittings are found to be consistent with predictions based on Goutennan's four orbital model. For horse heart and yeast cytochrome c, which show the greatest difference in the UV-visible band splittings, Stark effect measur ements on persistent spectral holes in the Q(0,0) band indicate that the pr otein-induced polarization is distinctly different for these two species. I ncorporation of the protein electrostatic field as virtual point charges in to quantum mechanical calculations utilizing the INDO/s semiempirical Hamil tonian is used to demonstrate that the effects of the protein on the heme e lectronic structure can be considerably different for the two proteins, con sistent with the experimental observations.