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

Isabelle Dragomir ^*, Andrew Hagarman ^*, Carmichael Wallace  and Reinhard Schweitzer-Stenner ^*

^* Department of Chemistry, Drexel University, Philadelphia, Pennsylvania; and Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

Correspondence: Address reprint requests to Reinhard Schweitzer-Stenner, Tel.: 215-895-2268, E-mail: rschweitzer-stenner{at}drexel.edu.

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 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 ironheme 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 d_xy-hole of Fe³⁺. For the ferro-state, the spectra resolve the vibronic structure of the Q_v-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 Q_v-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.