Conformational substates of horse heart cytochrome c exhibit different thermal unfolding of the heme cavity.
ABSTRACT The charge transfer (CT) band at 695 nm in the spectrum of ferri-cytochrome c is highly asymmetric, indicating conformational heterogeneity due to the coexistence of different conformational substates. We have measured the respective band profile of horse heart ferri-cytochrome c as a function of temperature between 283 K (10 degrees C) and 333 K (60 degrees C) and found that the well-known decrease of the absorptivity is wavenumber-dependent and exhibits a biphasic behavior. This indicates that the underlying conformational substates differ in their thermodynamic stability with respect to the structural changes associated with the disappearance of the 695 nm band, which eventually (at high temperatures) involves the replacement of M80 by a nearby lysine residue. Our data further indicates that the thermal unfolding process involves two structurally different intermediate states.
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ABSTRACT: Effect of anions of the Hofmeister series (thiocyanate, perchlorate, iodide, bromide, nitrate, chloride, sulfate, and phosphate) on local and global stability and flexibility of horse heart ferricytochrome c (cyt c) has been studied. Global stability of cyt c was determined by iso/thermal denaturations monitored by change in ellipticity in the far-UV region and its local stability was determined from absorbance changes in the Soret region. Particularly, relative stability/flexibility of the Met80-heme iron bond has been assessed by analysis of binding of cyanide into the heme iron. Both global and local stabilities of cyt c exhibited monotonous increase induced by a change of anion from chaotropic to kosmotropic species. However, this monotonous dependence was not observed for the rate constants of cyanide association with cyt c. As expected more chaotropic ions induced lower stability of protein and faster binding of cyanide but this correlation was reversed for kosmotropic anions. We propose that the unusual bell-shaped dependence of the rate constant of cyanide association is a result of modulation of Met80-heme iron bond strength and/or flexibility of heme region by Hofmeister anions independently on global stability of cyt c. Further, our results demonstrate sensitivity of cyanide binding to local change in stability/flexibility in the heme region of cyt c.Biophysical chemistry 07/2009; 144(1-2):21-6. · 2.28 Impact Factor
Article: Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular dichroism spectroscopy at cryogenic temperature.[show abstract] [hide abstract]
ABSTRACT: We have investigated the heterogeneity of the Fe(III)-Met80 linkage of horse heart ferricytochrome c by probing the 695nm 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 695nm band spectral heterogeneity, we have performed experiments as a function of pH (neutral and acidic) and temperature (room and 20K). 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 20K highlighted the heterogeneous nature of the 695nm 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 20K 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.Biophysical chemistry 12/2009; 147(1-2):8-12. · 2.28 Impact Factor
Article: Investigations on the Q and CT Bands of Cytochrome c Submonolayer Adsorbed on an Alumina Surface Using Broadband Spectroscopy with Single-Mode Integrated Optical Waveguides.[show abstract] [hide abstract]
ABSTRACT: In this work, we report experimental results on the molar absorptivity of cytochrome c adsorbed at different submonolayer levels onto an aluminum oxide waveguide surface; our data show a clear dependence of the protein optical properties on its surface density. The measurements were performed using the broadband, single-mode, integrated optical waveguide spectroscopic technique, which is an extremely sensitive tool able to reach submonolayer levels of detection required for this type of studies. This investigation focuses on the molar absorptivity at the Q-band (centered at 525 nm) and, for the first time to our knowledge, the weak charge transfer (CT) band (centered at 695 nm) of surface-adsorbed cyt c. Polarized light in the spectral region from 450 to 775 nm was all-coupled into an alumina thin film, which functioned as a single-mode planar optical waveguide. The alumina thin-film waveguide used for this work had a thickness of 180 nm and was deposited on a glass substrate by the atomic layer deposition process. The protein submonolayer was formed on the alumina waveguide surface through electrostatic adsorption from an aqueous buffer solution at neutral pH. The optical properties of the surface-adsorbed cyt c were investigated for bulk protein concentrations ranging from 5 nM to 8200 nM in the aqueous buffer solution. For a protein surface density of 2.3 pmol/cm(2), the molar absorptivity measured at the charge transfer band was 335 M(-1) cm(-1), and for a surface density of 15 pmol/cm(2) was 720 M(-1) cm(-1), which is much closer to the value of cyt c dissolved in an aqueous neutral buffer (830 M(-1) cm(-1)). The modification of the protein molar absorptivity and its dependence on the surface density can most likely be attributed to conformational changes of the surface-adsorbed species.The Journal of Physical Chemistry C 05/2009; 113(19):8306-8312. · 4.80 Impact Factor