Excited state lifetimes in cytochromes measured from Raman scattering data: evidence for iron-porphyrin interactions.

Abstract

ABSTRACr Resonance Raman scattering excitation profile data have been obtained on ferrocytochromes c and b5 in the a absorption band region. We observe in cytochrome c that the shape of the excitation profile agrees with the absorption band shape, while in cytochrome b5 it does not. In addition, we observe in cytochrome b5 a linewidth substantially larger than that in cytochrome c. From our data we conclude that the excited state lifetime in cytochrome c is longer than that in cytochrome b5 and that in cytochrome b5 the relaxation of the 1r-7r* excited state configuration of the porphyrin ring is different in the x direction than in the y direction. Possible origins of these effects due to coupling to the d-d transitions of the iron atom are discussed. strumentation utilizing dye laser sources to obtain tunability in the excitation frequency over the range of the a-band. To obtain the excitation profiles the intensities of the hemeprotein, Raman lines were compared to the intensity of either the ice Raman lines or the strong Raman line at 981 cm-1 of S042-[(NH4)2S04] that was added as a Raman intensity reference.Low-temperature absorption spectra were obtained by looking at the spectrum of white light scattered from the frozen sample and comparing it with the light scattered from a diffuse scatterer as well as by conventional transmission techniques using a Cary 15 spectrometer.The molecular and electronic properties of heme proteins have been heavily studied by a wide variety of techniques in an effort to understand on a molecular level the mechanisms of the oxidation-reduction processes. Most electronic structure information about the porphyrin macrocycle of these proteins has been obtained from the analysis of absorption (1) and fluorescence (2) data, but more recently several groups (refs. 3-10 and references cited in ref. 8) have applied the technique of resonance Raman scattering to these systems to determine molecular symmetry and electronic structure properties. In this study we have examined the well-known splitting of the a-absorption band in heme proteins by obtaining resonance Raman excitation profile data, i.e., the intensity of the Raman scattering as the excitation frequency is tuned through the absorption band. The a-bands of both ferrocytochrome c and ferrocytochrome bswere investigated. Our data demonstrate that the excited state lifetime resulting from the Qo,o transition (a-band) is shorter in cytochrome bs than it is in cytochrome c. They also show that in cytochrome b5 the excited state lifetimes of the split a-band differ substantially. Such a lifetime difference is absent in cytochrome c. We discuss possible explanations for these differences in terms of nonradiative relaxation via d-d transitions. METHODS AND MATERIALSCytochrome b5 was prepared from calves liver by the method of Omura and Takesue (11). It consists of protoporphyrin IX iron(II) in which the iron atom is bound to the protein through histidine ligands (12). Cytochrome c was purchased commercially (Sigma type VI). The iron ...

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

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