Modulation of the alkaline transition in cytochrome c and cytochrome c-T by full or specific partial acetimidylation.
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ABSTRACT:
SUBMITTER: Wallace CJ
PROVIDER: S-EPMC1153259 | biostudies-other | 1984 Feb
REPOSITORIES: biostudies-other
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Project description:Alkaline-induced conformational changes at pH 12.0 in the oxidized as well as the reduced state of cytochrome c oxidase have been systematically studied with time-resolved optical absorption and resonance Raman spectroscopies. In the reduced state, the heme a(3) first converts from the native five-coordinate configuration to a six-coordinate bis-histidine intermediate as a result of the coordination of one of the Cu(B) ligands, H290 or H291, to the heme iron. The coordination state change in the heme a(3) causes the alteration in the microenvironment of the formyl group of the heme a(3) and the disruption of the H-bond between R38 and the formyl group of the heme a. This structural transition, which occurs within 1min following the initiation of the pH jump, is followed by a slower reaction, in which Schiff base linkages are formed between the formyl groups of the two hemes and their nearby amino acid residues, presumably R38 and R302 for the heme a and a(3), respectively. In the oxidized enzyme, a similar Schiff base modification on heme a and a(3) was observed but it is triggered by the coordination of the H290 or H291 to heme a(3) followed by the breakage of the native proximal H378-iron and H376-iron bonds in heme a and a(3), respectively. In both oxidation states, the synchronous formation of the Schiff base linkages in heme a and a(3) relies on the structural communication between the two hemes via the H-bonding network involving R438 and R439 and the propionate groups of the two hemes as well as the helix X housing the two proximal ligands, H378 and H376, of the hemes. The heme-heme communication mechanism revealed in this work may be important in controlling the coupling of the oxygen and redox chemistry in the heme sites to proton pumping during the enzymatic turnover of CcO.
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Project description:We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pH-induced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0-9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5-10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry.
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Project description:Magnetic-circular-dichroism (m.c.d.) spectra over the wavelength range 300-2000 nm at room temperature and at 4.2K of horse heart cytochrome c are reported at a series of pH values between 7.8 and 11.0, encompassing the alkaline transition. The effect of glassing agents on the e.p.r. spectrum at various pH values is also reported. Comparison of these results with spectra obtained for the n-butylamine adduct of soybean leghaemoglobin support the hypothesis that lysine is the sixth ligand in the alkaline form of horse heart cytochrome c. The m.c.d. and e.p.r. spectra of horse heart cytochrome c in the presence of 1-methylimidazole have also been examined. These studies strongly suggest that histidine-18, the proximal ligand of the haem, is the ionizing group that triggers the alkaline transition. Low-temperature m.c.d. and e.p.r. spectra are also reported for Pseudomonas aeruginosa cytochrome c551. It is shown that no ligand exchange takes place at the haem in this species over the pH range 6.0-11.3.
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Project description:The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021).
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Project description:Alkaline conformers of cytochrome c may be involved in both its electron transport and apoptotic functions. We use cobalt(II)bis(terpyridine), Co(terpy)2(2+), as a reagent for conformationally gated electron-transfer (gated ET) experiments to study the alkaline conformational transition of K79H variants of yeast iso-1-cytochrome c expressed in Escherichia coli , WT*K79H, with alanine at position 72 and Saccharomyces cerevisiae , yK79H, with trimethyllysine (Tml) at position 72. Co(terpy)2(2+) is well-suited to the 100 ms to 1 s time scale of the His79-mediated alkaline conformational transition of these variants. Reduction of the His79-heme alkaline conformer by Co(terpy)2(2+) occurs primarily by gated ET, which involves conversion to the native state followed by reduction, with a small fraction of the His79-heme alkaline conformer directly reduced by Co(terpy)2(2+). The gated ET experiments show that the mechanism of formation of the His79-heme alkaline conformer involves only two ionizable groups. In previous work, we showed that the mechanism of the His73-mediated alkaline conformational transition requires three ionizable groups. Thus, the mechanism of heme crevice opening depends upon the position of the ligand mediating the process. The microscopic rate constants provided by gated ET studies show that mutation of Tml72 (yK79H variant) in the heme crevice loop to Ala72 (WT*K79H variant) affects the dynamics of heme crevice opening through a small destabilization of both the native conformer and the transition state relative to the His79-heme alkaline conformer. Previous pH jump data had indicated that the Tml72?Ala mutation primarily stabilized the transition state for the His79-mediated alkaline conformational transition.
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Project description:The biological consequences of acetimidylation of all 19 epsilon-amino groups of horse cytochrome c are a slight decrease in both the redox potential of the protein and its ability to stimulate oxygen uptake in the cytochrome c-depleted-mitochondria assay. Examination of a number of specific partially acetimidylated analogues and acetimidylated cytochromes c of other species has shown that the changes in biological properties, which are associated with a slight structural change as monitored by n.m.r. spectroscopy [Boswell, Moore, Williams, Harris, Wallace, Bocieck & Welti (1983) Biochem. J. 213, 679-686], appear to stem from modification of residues in a restricted region of the sequence. The failure of the redox potential of Saccharomyces cerevisae cytochrome c to be affected by acetimidylation suggests that it is lysine-53, absent from that species, that is the sensitive residue.
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