A. M. Tamburro

Cytochrome c interaction with the mitochondrial membrane: A spin label study

Abstract Horse-heart ferrocytochrome c has been labeled with N -(2,2,5,5-tetramethyl-3-pyrrolidinyl-1-oxyl) iodoacetamide at methionine-65. The paramagnetic resonance spectrum of labeled ferricytochrome c indicates a weak immobilization of the radical ( τ c = 9.3·10 −10 sec) which becomes stronger upon binding of labeled cytochrome c to cytochrome c -depleted mitochondrial membranes ( τ c = 3.3·10 −9 sec). The hyperfine coupling constant remains, however, unchanged (16.7 ± 0.1 gauss) indicating that the cytochrome c binding site is highly polar. The region where cytochrome c is bound to the membrane is insensitive to large variations of medium viscosity.

Photooxidative and spectral studies on cytochrome c. Conformational changes induced by binding of cardiolipin.

Abstract— Binding of cardiolipin to ferrocytochrome c, to form a 4:1 molar complex, results in an approximately sixfold increase of the tryptophan fluorescence emission. Furthermore, appreciable perturbations of the circular dichroism spectrum occur in the Soret and in the far‐ultraviolet regions. The irradiation of the ferrocytochrome–cardiolipin complex at pH 8·8 with visible light leads to the photooxidative modification of histidine‐18, tyrosine‐48 and methionine‐80. Comparison of the above findings with those concerning unbound ferrocytochrome c suggests that the interaction between cardiolipin and ferrocytochrome c provokes a perturbation of the protein conformation, which possibly involves the disruption of the hydrogen bonds linking the aromatic rings of tryptophan‐59 and tyrosine‐48 with one propionic side chain of the heme.

Photo-oxidative modification of the heme ligands in horse heart ferricytochrome c: Conformational and functional studies

Abstract The selective photo-oxidation of either histidine-18 or methionine-80 of horse heart ferricytochrome c has been achieved. The histidine-18 modified cytochrome exhibits no enzymic activity, despite limited conformational changes with respect to the native protein. The activity of the methionine-80 modified cytochrome is instead almost fully maintained, coordination with the heme iron being still possible through the sulfoxide formed by photo-oxidation of methionine thioether. On the other hand, this derivative, while maintaining a rigid spatial orientation of the heme, undergoes large conformational rearrangements. The discrepancy between structural and functional data can be explained by a lack of specific conformational requirements for cytochrome c activity. Alternatively, a specific conformation necessary for the activity of the protein may be induced by cytochrome-membrane interaction.

Proteomics for the discovery of nuclear bile acid receptor FXR targets

Nuclear receptors (NRs) are important pharmacological targets for a number of diseases, including cancer and metabolic disorders. To unmask the direct role of NR function it is fundamental to find the NR targets. During the last few years several NRs have been shown to affect microRNA expression, thereby modulating protein levels. The farnesoid X receptor (FXR), the main regulator of bile acid (BA) homeostasis, also regulates cholesterol, lipid and glucose metabolism. Here we used, for the first time, a proteomics approach on mice treated with a FXR ligand to find novel hepatic FXR targets. Nineteen spots with a more than two-fold difference in protein amounts were found by 2D-DIGE and 20 proteins were identified by MALDI-TOF MS as putative novel FXR targets. The most striking feature of the protein list was the great number of mitochondrial proteins, indicating a substantial impact of FXR activation on mitochondrial function in the liver. To examine if the differences found in the proteomics assay reflected differences at the mRNA level, a microarray assay was generated on hepatic samples from wild type and FXR−/− mice treated with a FXR ligand and compared to vehicle treatment. At least six proteins were shown to be regulated only at a post-transcriptional level. In conclusion, our study provides the impetus to include proteomic analysis for the identification of novel targets of transcription factors, such as NRs. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.