Optical Band Splitting and Electronic Perturbations of the Heme Chromophore in Cytochrome c at Room Temperature Probed by Visible Electronic Circular Dichroism Spectroscopy

Abstract

We have measured the electronic circular dichroism (ECD) of the ferri- and ferro-states of several natural cytochrome c derivatives (horse heart, chicken, bovine, and yeast) and the Y67F mutant of yeast in the region between 300 and 750 nm. Thus, we recorded the ECD of the B- and Q-band region as well as the charge-transfer band at approximately 695 nm. The B-band region of the ferri-state displays a nearly symmetric couplet at the B0-position that overlaps with a couplet 790 cm-1 higher in energy, which we assigned to a vibronic side-band transition. For the ferro-state, the couplet is greatly reduced, but still detectable. The B-band region is dominated by a positive Cotton effect at energies lower than B0 that is attributed to a magnetically allowed iron-->heme charge-transfer transition as earlier observed for nitrosyl myoglobin and hemoglobin. The Q-band region of the ferri-state is poorly resolved, but displays a pronounced positive signal at higher wavenumbers. This must result from a magnetically allowed transition, possibly from the methionine ligand to the dxy-hole of Fe3+. For the ferro-state, the spectra resolve the vibronic structure of the Qv-band. A more detailed spectral analysis reveals that the positively biased spectrum can be understood as a superposition of asymmetric couplets of split Q0 and Qv-states. Substantial qualitative and quantitative differences between the respective B-state and Q-state ECD spectra of yeast and horse heart cytochrome c can clearly be attributed to the reduced band splitting in the former, which results from a less heterogeneous internal electric field. Finally, we investigated the charge-transfer band at 695 nm in the ferri-state spectrum and found that it is composed of at least three bands, which are assignable to different taxonomic substates. The respective subbands differ somewhat with respect to their Kuhn anisotropy ratio and their intensity ratios are different for horse and yeast cytochrome c. Our data therefore suggests different substate populations for these proteins, which is most likely assignable to a structural heterogeneity of the distal Fe-M80 coordination of the heme chromophore.

Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular dichroism spectroscopy at cryogenic temperature

“…This spectrum is very well resolved: it shows a shoulder at ~14250 cm -1 , a peak at ~14500 cm -1 , and a clearly resolved peak at -1 ; as for the pH 7 sample, the high frequency wing is characterized by peaks at approximately 15400 and 15800 cm -1 . Note that the small band at about 13650 cm -1 reported by Dragomir et al (23) and named S1 by the authors, is never observed in our experiments (both in absorption and ECD) even at 20 K.…”

“…Following the idea of Schweitzer-Stenner and coworkers (23,28,30), we complemented the absorption data with ECD data on the very same samples. The ECD spectrum of ferricytochrome c at 20 K and pH 7 ( Figure 2d) shows an asymmetric peak at ~14500 cm -1 and a clearly resolved peak at ~15000 cm -1 ; the high frequency wing of the spectrum is characterized by a positive peak a ~15400 cm -1 and negative one at ~15800 cm -1 .…”

Cytochrome c: A Multifunctional Protein Combining Conformational Rigidity with Flexibility

“…More direct evidence for -band splitting was recently provided by us [37,81] based on a comparison of visible CD and absorption profiles in the Soret band region. Figure 10 depicts CD and absorption in the Soret band region of ferriand ferrocytochrome c. The CD spectrum of the oxidized protein shows a clear couplet, whereas the corresponding spectrum of the reduced species is somewhat more complex.…”

“…However, the same effect does not imply the same cause. First of all, a sole inspection of the CD spectrum does not reveal all necessary facts; one has to compare CD with their corresponding absorption spectra [81,82]. The absence of the negative couplet in the CD spectra of the above discussed F87 mutants of yeast ferricytochrome c could just reflect the elimination of one important mode of transition dipole coupling between protein and heme, which contributes to the negative rotational strength of one of the -state transitions.…”

“…However a comparison of CD and absorption spectra reveals a noncoincidence at the 0 -position which indicates band splitting ( Figure 11) [37,81]. Moreover, the CD spectra show a much better resolution of the vibronic V -band which facilitates the analysis further.…”