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

Abstract

We have investigated the heterogeneity of the Fe(III)–Met80 linkage of horse heart ferricytochrome c by probing the 695 nm charge transfer band with absorption and electronic circular dichroism (ECD) spectroscopy. In order to verify the connection between conformational substates of the Fe(III)–Met80 linkage and the 695 nm band spectral heterogeneity, we have performed experiments as a function of pH (neutral and acidic) and temperature (room and 20 K). At room temperature, the ECD spectrum is blue shifted with respect to the absorption one; the shift is more pronounced at acidic pH and is compatible with the presence of sub-bands. ECD measurements at 20 K highlighted the heterogeneous nature of the 695 nm band and provided direct experimental evidence for the presence of sub-bands. Indeed, while the absorption spectra remained deceivingly unstructured, the ECD spectra showed well resolved peaks and shoulders. A consistent fit of the 20 K absorption and ECD spectra showed that five Gaussians (each centered at the same frequency in the absorption and ECD spectrum) are able to reproduce the observed lineshapes. A careful analysis of frequency shifts and intensity ratios of these sub-bands enabled us to identify at least three distinct sub-bands arising from taxonomic conformational substates of the Fe(III)–Met80 linkage. In view of the major influence of the Fe(III)–Met80 linkage on the redox potential of ferricytochrome c, we speculate that these spectrally distinguishable substates may have different functional roles

Cytochrome c: A Multifunctional Protein Combining Conformational Rigidity with Flexibility

“…We had shown earlier that the 695 nm band can be decomposed into different subbands that should be related to different substates of the Fe-M80 linkage [81]. Our analysis was later confirmed by the results of low temperature CD measurements [131]. As recognized by Frauenfelder and coworkers, proteins in different subconformations perform the same function but with different rates [132].…”

Insights into Structure and Function of the Active Site of SoxAX Cytochromes

“…Orientations of the side chains of Arg 232 and Gln 239 and the heme propionate groups also differ in chains A and C, which further suggests that the heme environment in the SoxA subunit of SnSoxAX C236M has some inherent flexibility. MCD Spectroscopy-The UV-visible MCD spectra of SnSoxAX C236M show intense bands characteristic of low spin ferric heme, including a sharp negative feature at ϳ690 nm characteristic of a sulfur to Fe charge transfer (CT) transition p(S) 3 d p (Fe) in His/Met ligated hemes (39,40) that is also seen in SnSoxAX WT spectra (data not shown). In the 1000 -2200 nm region, porphyrin to iron CT transitions with energies diagnostic of the axial ligation (41,42) (Fig.…”

A mitokondriális citokróm c poszttranszlációs érése

“…A 695 nm-es csúcs hiánya nem jelenti minden esetben a ligandum hiányát. A csúcs eltűnhet akkor is, ha a 80-as metionin pozíciója a hem síkjához képest kissé megváltozik (Ångström 1982), de úgyszintén, ha a hem zseb kisebb konformációs változást szenved (Spilotros 2010).…”