Increasing the redox potential of isoform 1 of yeast cytochrome c through the modification of select haem interactions

2002 ◽  
Vol 362 (2) ◽  
pp. 281-287 ◽  
Author(s):  
C. Marc LETT ◽  
J. Guy GUILLEMETTE
Keyword(s):  
Cytochrome C ◽  
Redox Potential ◽  
Point Mutations ◽  
Cd Spectroscopy ◽  
Wild Type ◽  
Mutant Proteins ◽  
Oxidation Reduction ◽  
Oxidation Reduction Potential ◽  
Biophysical Studies ◽  
Cytochromes C

The oxidation—reduction potential of eukaryotic cytochromes c varies very little from species to species. We have introduced point mutations into isoform 1 of yeast cytochrome c (iso-1-cytochrome c) to selectively engineer a protein with a higher redox potential. Of the ten different mutant proteins generated for the present investigation Y67R, Y67K and W59H were found to be non-functional. Three other mutant proteins, L32M, L32T and T49K, were functional, but too unstable for biophysical studies. Mutant cytochromes c K79S, K79T, Y48H and Y48K were purified and characterized. The Y48K mutant is the only one that exhibits a significant increase of +117mV in redox potential compared with the wild-type protein while still supporting oxidative phosphorylation invivo. Low temperature difference spectroscopy confirmed the formation of the holoprotein, while adsorption and CD spectroscopy reveal perturbations in the structure of Y48K iso-1-cytochrome c.

scholarly journals The effect of complete or specific partial acetimidylation on the biological properties of cytochrome c and cytochrome c-T

1984 ◽  
Vol 217 (3) ◽  
pp. 595-599 ◽  
Author(s):  
C J A Wallace
Keyword(s):  
Structural Change ◽  
Cytochrome C ◽  
Oxygen Uptake ◽  
Redox Potential ◽  
Biological Properties ◽  
Amino Groups ◽  
Cytochromes C ◽  
Restricted Region ◽  
Horse Cytochrome

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.

scholarly journals S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) ReduceIn VitroInhibition by Fluconazole

2012 ◽  
Vol 56 (4) ◽  
pp. 2099-2107 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Jonathan G. L. Mullins ◽  
Claire M. Hull ◽  
Josie E. Parker ◽  
David C. Lamb ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Point Mutations ◽  
Triazole Ring ◽  
Wild Type ◽  
Protein Activity ◽  
Content Type ◽  
Mutant Proteins ◽  
Type Protein ◽  
Wild Type Protein ◽  
Binding Spectra

ABSTRACTThe effects of S279F and S279Y point mutations inCandida albicansCYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (Ks, 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (Ks, 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC50s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higherKdvalues observed.

Control of oxidation-reduction potential during Cheddar cheese ripening and its effect on the production of volatile flavour compounds

2016 ◽  
Vol 83 (4) ◽  
pp. 479-486 ◽  
Author(s):  
Veronica Caldeo ◽  
John A Hannon ◽  
Dara-Kate Hickey ◽  
Dave Waldron ◽  
Martin G Wilkinson ◽  
...  
Keyword(s):  
Redox Potential ◽  
Cheddar Cheese ◽  
Principal Component ◽  
Reducing Agents ◽  
Sulphur Compounds ◽  
Oxidation Reduction ◽  
Oxidation Reduction Potential ◽  
Volatile Flavour ◽  
Redox Agents ◽  
The Stability

In cheese, a negative oxidation-reduction (redox) potential is required for the stability of aroma, especially that associated with volatile sulphur compounds. To control the redox potential during ripening, redox agents were added to the salted curd of Cheddar cheese before pressing. The control cheese contained only salt, while different oxidising or reducing agents were added with the NaCl to the experimental cheeses. KIO3 (at 0·05, 0·1 and 1%, w/w) was used as the oxidising agent while cysteine (at 2%, w/w) and Na2S2O4 (at 0·05 and 0·1%, w/w) were used as reducing agents. During ripening the redox potential of the cheeses made with the reducing agents did not differ significantly from the control cheese (Eh ≈ −120 mV) while the cheeses made with 0·1 and 0·05% KIO3 had a significantly higher and positive redox potential in the first month of ripening. Cheese made with 1% KIO3 had positive values of redox potential throughout ripening but no starter lactic acid bacteria survived in this cheese; however, numbers of starter organisms in all other cheeses were similar. Principal component analysis (PCA) of the volatile compounds clearly separated the cheeses made with the reducing agents from cheeses made with the oxidising agents at 2 month of ripening. Cheeses with reducing agents were characterized by the presence of sulphur compounds whereas cheeses made with KIO3 were characterized mainly by aldehydes. At 6 month of ripening, separation by PCA was less evident. These findings support the hypothesis that redox potential could be controlled during ripening and that this parameter has an influence on the development of cheese flavour.

scholarly journals Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

1994 ◽  
Vol 302 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A Schejter ◽  
T I Koshy ◽  
T L Luntz ◽  
R Sanishvili ◽  
I Vig ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
General Increase ◽  
Reduction Potentials ◽  
Cytochromes C ◽  
In The Wild ◽  
Order Of Magnitude ◽  
The Stability ◽  
Haem Iron

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

scholarly journals PLP1 Mutations in Patients with Multiple Sclerosis: Identification of a New Mutation and Potential Pathogenicity of the Mutations

2018 ◽  
Vol 7 (10) ◽  
pp. 342 ◽  
Author(s):  
Nancy Cloake ◽  
Jun Yan ◽  
Atefeh Aminian ◽  
Michael Pender ◽  
Judith Greer
Keyword(s):  
Multiple Sclerosis ◽  
Clinical Symptoms ◽  
Point Mutations ◽  
Human Leukocyte ◽  
In Silico Analysis ◽  
Proteolipid Protein ◽  
Wild Type ◽  
Exon 2 ◽  
Mutant Proteins ◽  
The Unfolded Protein Response

PLP1 is located on the X-chromosome and encodes myelin proteolipid protein (PLP), the most abundant protein in central nervous system myelin. Generally, point mutations in PLP1 result in X-linked dysmyelinating disorders, such as Pelizaeus-Merzbacher disease (PMD) or spastic paraplegia type 2 (SPG2). However, several case studies have identified patients with missense point mutations in PLP1 and clinical symptoms and signs compatible with a diagnosis of multiple sclerosis (MS). To investigate if PLP1 mutations occur relatively frequently in MS, we sequenced the coding regions of PLP1 in 22 female MS patients who had developed disease after the age of 40 and in 42 healthy women, and identified a missense mutation in exon 2 of PLP1 resulting in a Leu30Val mutation in the protein in one of the MS patients. mCherry-tagged plasmids containing wild type or mutant PLP1 sequences of PLP, including two known PMD/SPG2-related mutations as positive controls, were constructed and transfected into Cos-7 cells. In comparison with cells transfected with wild type PLP1, all mutations caused significant accumulation of PLP in the endoplasmic reticulum of the cells and induction of the unfolded protein response—a mechanism that leads to apoptosis of cells expressing mutant proteins. Additionally, in silico analysis of the binding of peptides containing the Leu30Val mutation to the human leukocyte antigen (HLA) molecules carried by the patient harboring this mutation suggested that the mutation could produce several novel immunogenic epitopes in this patient. These results support the idea that mutations in myelin-related genes could contribute to the development of MS in a small proportion of patients.

scholarly journals Studies on the biosynthesis of cytochrome c

1973 ◽  
Vol 134 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Emer M. Colleran ◽  
O. T. G. Jones
Keyword(s):  
Cytochrome C ◽  
Specific Radioactivity ◽  
Intermediate Formation ◽  
Maximum Deviation ◽  
Midpoint Potential ◽  
Oxidation Reduction ◽  
High Specific Radioactivity ◽  
Absolute Requirement ◽  
Mammalian Mitochondria ◽  
Cytochromes C

A soluble cytochrome was isolated and purified from the slime mould Physarum polycephalum and identified as cytochrome c by room-temperature and low-temperature (77°K) difference spectroscopy. A close similarity between P. polycephalum and mammalian cytochromes c was suggested by a comparison of the initial rates of oxidation of both proteins by mammalian mitochondria. This similarity was further emphasized by redox titrations and gel-electrophoretic studies which indicated that P. polycephalum cytochrome c has an oxidation–reduction midpoint potential of +257mV at pH7.0 and a molecular weight of 12500±1500 (mean±maximum deviation for a set of six measurements). P. polycephalum exhibits an absolute requirement for protohaemin for growth. The 59Fe-labelled haemin was prepared by chemical synthesis from protoporphyrin. The purified product had a specific radioactivity of 0.8±0.02μCi/mol. Growth of P. polycephalum in the presence of [59Fe]haemin resulted in the incorporation of 59Fe into the plasmodial cytochrome c. The specific radioactivity of the cytochrome c haem was 0.36±0.02μCi/mol. The high specific radioactivity of the cytochrome haem indicates that synthesis of the holoenzyme must proceed by direct attachment of haem to the apoprotein rather than by the intermediate formation of a protoporphyrinogen–apoprotein complex. The observed decrease in the specific radioactivity of the haem group is attributed to exchange of the 59Fe with unlabelled iron in the plasmodia either before or during attachment of the haem group to the apoprotein.

scholarly journals Mutations in the N- and C-terminal Tails of Potato Carboxypeptidase Inhibitor Influence Its Oxidative Refolding Process at the Reshuffling Stage

2001 ◽  
Vol 276 (15) ◽  
pp. 11683-11690 ◽  
Author(s):  
Gabriela Venhudová ◽  
Francesc Canals ◽  
Enrique Querol ◽  
Francesc X. Aviles
Keyword(s):  
Noncovalent Interactions ◽  
Point Mutations ◽  
Second Phase ◽  
Wild Type ◽  
Lower Efficiency ◽  
Native Form ◽  
Mutant Proteins ◽  
Oxidative Refolding ◽  
Stable Species ◽  
Carboxypeptidase Inhibitor

A comparative study of the oxidative refolding for nine selected potato carboxypeptidase inhibitor (PCI) mutants was carried out using the disulfide quenching approach. The mutations were performed at the N- and C-terminal tails of PCI outside its disulfide stabilized central core. The differences between the refolding of wild type and mutant proteins were observed in the second phase of the refolding process, the reshuffling of disulfide bridges, although the first phase, nonspecific packing, was not greatly affected by the mutations. Point mutations at the C-tail or deletion of up to three C-terminal residues of PCI resulted in a lower efficiency of the reshuffling process. In the case of the mutants lacking five N-terminal or four or five C-terminal residues, no “native-like” form was observed after the refolding process. On the other hand, the double mutant G35P/P36G did not attain a native-like form either, although one slightly more stable species was observed after being submitted to refolding. The disulfide pairing of this species is different from that of the wtPCI native form. The differences between the refolding process of wild type and mutant forms are interpreted in the light of the new view of protein folding. The results of the present study support the hypothesis that the refolding of this small disulfide-rich protein, and others, is driven by noncovalent interactions at the reshuffling stage. It is also shown that the interactions established between the N- and C-tail residues and the core of PCI are important for the proper refolding of the protein.

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