Conformational Substates of Horse Heart Cytochrome <i>c</i> Exhibit Different Thermal Unfolding of the Heme Cavity

Schweitzer‐Stenner, Reinhard; Shah, Ronak; Hagarman, and Andrew M.; Dragomir, Isabelle

doi:10.1021/jp069022j

Cited by 25 publications

(26 citation statements)

References 29 publications

(71 reference statements)

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“…This indicates that our experimental conditions of buffer solution concentration (0.1 M PBS) kept substantially almost unaltered the heme surrounding conformation and therefore the protein is not denaturalised at pH 2.0. 28,29 As shown above, with the increase of the solution pH from 2.0 to 8.0, E 0 0 shied very slightly to lower values giving a variation of 65 mV. Moreover, it is remarkable that the peak current ratio (I pc /I pa )extracted from Fig.…”

Section: Inuence Of Solution Ph Value On the Electrochemistry Of Cyt...mentioning

confidence: 59%

Screen-printed graphite macroelectrodes for the direct electron transfer of cytochrome c: a deeper study of the effect of pH on the conformational states, immobilization and peroxidase activity

Gómez-Mingot

Montiel

Banks

et al. 2014

Analyst

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The direct electron transfer of cytochrome c has been studied at screen-printed graphite macroelectrodes without recourse to mediators or the need for any electrode pre-treatment as is commonly employed within the literature. A wide range of pH values from 2.0 to 11.0 have been explored upon the electrochemical response of cytochrome c and different voltammetric signatures have been observed. The direct electron transfer of the alkaline transition of cytochrome c was found impeded within alkaline media leading to either an irreversible redox process or even no voltammetric responses. In acidic aqueous media the electrochemical process is observed to undergo a mixed diffusion and adsorption controlled process rather than a purely diffusional process of the native conformation as observed at pH 7.0. Interestingly, at pH 3.5 a new conformational state is revealed in cooperation with the native conformation. The immobilization of the protein was satisfactorily obtained using a simple method by cycling the protein at specific solution pH values allowing amperometric responses to be obtained and gives rise to useful pseudo-peroxidase activity for sensing H2O2. Apparent Michaelis-Menten constant values (Km) were calculated via the Lineweaver-Burk method with deduced values of 25 ± 4, 98 ± 12 and 230 ± 30 mM, respectively for pH values of 2.0, 3.0 and 7.0. Such work is important for those utilising cytochrome c in bio-electrochemical and related applications.

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Section: Inuence Of Solution Ph Value On the Electrochemistry Of Cyt...mentioning

confidence: 59%

Screen-printed graphite macroelectrodes for the direct electron transfer of cytochrome c: a deeper study of the effect of pH on the conformational states, immobilization and peroxidase activity

Gómez-Mingot

Montiel

Banks

et al. 2014

Analyst

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“…In this respect, we note that contrary to its ferrous counterpart that exhibits a high thermal stability, requiring temperatures above 100 °C (373 K) to undergo unfolding, 70 ferric Cyt c at pH7 starts unfolding at considerably lower temperatures of 40 °C (313 K) to 70 °C (343 K), going through different unfolding states. [71][72][73] While resonance Raman spectra indicate that the LS state of the haem iron is maintained in some of these states, 74 it is unclear to what extent the remaining conformational transitions are related to higher spin states and whether these have a functionally relevant role.…”

Section: Resultsmentioning

confidence: 99%

Spin cascade and doming in ferric hemes: Femtosecond X-ray absorption and X-ray emission studies

Bacellar

Kinschel

Mancini

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The structure–function relationship is at the heart of biology, and major protein deformations are correlated to specific functions. For ferrous heme proteins, doming is associated with the respiratory function in hemoglobin and myoglobins. Cytochrome c (Cyt c) has evolved to become an important electron-transfer protein in humans. In its ferrous form, it undergoes ligand release and doming upon photoexcitation, but its ferric form does not release the distal ligand, while the return to the ground state has been attributed to thermal relaxation. Here, by combining femtosecond Fe Kα and Kβ X-ray emission spectroscopy (XES) with Fe K-edge X-ray absorption near-edge structure (XANES), we demonstrate that the photocycle of ferric Cyt c is entirely due to a cascade among excited spin states of the iron ion, causing the ferric heme to undergo doming, which we identify. We also argue that this pattern is common to a wide diversity of ferric heme proteins, raising the question of the biological relevance of doming in such proteins.

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“…The charge transfer band has long been hailed as a marker of the integrity of the Fe–S bond of cytochrome c 34 and, thus, a probe for protein conformational changes 20. However, it is too weak for conventional instrumentation to corroborate the aforementioned changes in the Q-band.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2009

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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.

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Conformational Substates of Horse Heart Cytochrome c Exhibit Different Thermal Unfolding of the Heme Cavity

Cited by 25 publications

References 29 publications

Screen-printed graphite macroelectrodes for the direct electron transfer of cytochrome c: a deeper study of the effect of pH on the conformational states, immobilization and peroxidase activity

Screen-printed graphite macroelectrodes for the direct electron transfer of cytochrome c: a deeper study of the effect of pH on the conformational states, immobilization and peroxidase activity

Spin cascade and doming in ferric hemes: Femtosecond X-ray absorption and X-ray emission studies

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

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