- Figure 1: X-ray absorption spectra. (a) Overlay of the X-ray absorption spectra for reduced WT (red) and Y48pCMF (green). Insert: detailed view highlighting the differences within the XANES region. (b) EXAFS extracted signals. (c) Overlay of the Fourier transforms of the EXAFS spectra. In panels (b) and (c), continuous lines correspond to experimental data and dotted lines to theoretical fits.
The response of cells to physiological changes and oxidative stress involves the modulation of mitochondrial respiration. This affects the intrinsic activities of the electron transport chain components and their assembly within the internal mitochondrial membrane. In fact, hypoxia induces the grouping of these proteins into the so-called supercomplexes, which facilitates the transfer of electrons between the distinct components of the electron transport chain, while decreasing the generation of reactive oxygen species (ROS). The best known supercomplex is called the respirasome, and encompasses the membrane complexes I, III and IV. Alterations in the formation of the supercomplex lead to the development of hypoxia-dependent pathologies, such as cancer or ischemia. A key modulator of the mitochondrial activity is cytochrome c (Cc)1. Cc is a small heme-protein which acts as an electron carrier between complexes III and IV; and is highly conserved throughout evolution. Cc also participates in cell life and death decisions in mammals2 and plants3. This pleiotropic role of Cc makes post-translational modifications an essential mechanism to regulate its functions tightly. Phosphorylation and nitration of tyrosine residues stand out among these modifications. Indeed, they affect the conformational equilibria of the protein along with its ability to bind physiological partners3,4.
Specifically, post-translational Cc phosphorylation at tyrosine 48 is key in modulating mitochondrial signalling. Nevertheless, the mechanism by which it alters the conformation and function of this protein is barely understood. To address this subject requires solving the 3D structure of the phosphorylated species. However, cell-extract phosphatases reverse Cc phosphorylation, hampering the isolation of the modified protein from tissues. This makes any structural analysis a highly challenging task. Hence, we solved the 3D solution-structure of a Cc phosphomimic obtained by replacing tyrosine 48 with the synthetic amino acid p-carboxy-methyl-L-phenylalanine (pCMF)5.
In order to do this, the effects of the mutation on the heme iron coordination sphere were tested. First, distance restraints relative to the axial coordination of iron were necessary for the structure computations. All the reported mutants mimicking phosphorylation in this position displayed altered biophysical properties related to conformational stability of the heme moiety3,5. Thus, a precise assessment of the changes in coordination geometry of the heme iron was required. Figure 1a shows the XAS fluorescence spectra at the Fe K-edge of wild-type (WT) and mutant (Y48pCMF) Cc species at cryogenic temperatures, recorded at the I20-Scanning beamline. Notably, the absorption spectra of the two proteins are very alike. Both spectra include the pre-edge feature at ca. 7113 eV, indicative of the octahedral geometry for the first iron coordination sphere. Overall, the X-ray Absorption Near Edge Structure (XANES) region of the spectra for the two Cc species are almost identical, as can be seen in the figure. The absorption edge appears at 7126 eV in both cases, indicating that the oxidation state of the metal ion is not affected by the mutation. However, some spectral features are broadened for the Y48pCMF Cc, as is the case of the feature at 7136 eV, related to porphyrin plane distortion.