Stability Of Cytochrome Research Articles

Suitability of Adenosine Derivatives in Improving the Activity and Stability of Cytochrome c under Stress: Insights into the Effect of Phosphate Groups.

It is well known that adenosine and its phosphate derivatives play a crucial role in biological phenomena such as apoptosis and cell signaling and act as the energy currency of the cell. Although their interactions with various proteins and enzymes have been described, the focus of this work is to demonstrate the effect of the phosphate group on the activity and stability of the native heme metalloprotein cytochrome c (Cyt c), which is important from both biological and industrial aspects. In situ and in silico characterizations are used to correlate the relationship between the binding affinity of adenosine and its phosphate groups with unfolding behavior, corresponding peroxidase activities, and stability factors. Interaction of adenosine (ADN), adenosine monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP) with Cyt c increases peroxidase-like activity by up to 1.8-6.5-fold compared to native Cyt c. This activity is significantly maintained even after multiple stress conditions such as oxidative stress and the presence of a chaotropic agent such as guanidine hydrochloride (GuHCl). With binding affinities on the order of ADN < AMP < ADP < ATP, adenosine derivatives were found to stabilize Cyt c by varying the secondary structural features of the protein. Thus, in addition to being a fundamental study, the current work also proposes a way of stabilizing protein systems to be used for real-time biocatalytic applications.

Soft interactions versus hard core repulsions: A journey of cytochrome c from acid-induced denaturation to native protein via pre-molten globule and molten globule conformations exploiting dextran and its monomer glucose

Protein attains its biological functional state in a crowded environment, whereas traditional in vitro experiments are carried out in dilute solutions. In this investigation, synthetic crowders (dextran and glucose) are exploited to investigate the effect of crowding on the conformation of cytochrome c. Protein folding may occur through many non-native intermediate states. Here, the effect of crowder (dextran) and its monomer (glucose) on the structural conformation and stability of cytochrome c (cyt c) has been studied in an acidic environment using various biophysical techniques such as UV–Vis absorbance, circular dichroism, and fluorescence spectroscopy (Trp and ANS fluorescence), dynamic light scattering and interaction experiments using ITC and molecular docking. These techniques show that the cyt c forms a molten globule in the presence of glucose while the pre-molten globule in the presence of the dextran 70. These results suggest that dextran 70 stabilizes the protein due to macromolecular crowding via soft interactions, while glucose due to co-solute engineering exploiting volume exclusion phenomenon as confirmed by ITC experiments. These investigations inferred that crowders protect protein in harsh conditions (acidic pH), which may arise due to various diseased conditions such as acidosis-induced insulin resistance, cardiovascular risks, etc. The current work also has an industrial application to increase the shelf life of therapeutic proteins.

A genome-wide copper-sensitized screen identifies novel regulators of mitochondrial cytochrome c oxidase activity

Copper is essential for the activity and stability of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Loss-of-function mutations in genes required for copper transport to CcO result in fatal human disorders. Despite the fundamental importance of copper in mitochondrial and organismal physiology, systematic identification of genes that regulate mitochondrial copper homeostasis is lacking. To discover these genes, we performed a genome-wide screen using a library of DNA-barcoded yeast deletion mutants grown in copper-supplemented media. Our screen recovered a number of genes known to be involved in cellular copper homeostasis as well as genes previously not linked to mitochondrial copper biology. These newly identified genes include the subunits of the adaptor protein 3 complex (AP-3) and components of the cellular pH-sensing pathway Rim20 and Rim21, both of which are known to affect vacuolar function. We find that AP-3 and Rim mutants exhibit decreased vacuolar acidity, which in turn perturbs mitochondrial copper homeostasis and CcO function. CcO activity of these mutants could be rescued by either restoring vacuolar pH or supplementing growth media with additional copper. Consistent with these genetic data, pharmacological inhibition of the vacuolar proton pump leads to decreased mitochondrial copper content and a concomitant decrease in CcO abundance and activity. Taken together, our study uncovered novel genetic regulators of mitochondrial copper homeostasis and provided a mechanism by which vacuolar pH impacts mitochondrial respiration through copper homeostasis.

Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

Stability of cytochromes c' from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure.

The stability of dimeric cytochrome c' from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit-subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c' from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c' from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure.

Mutations in the UQCC1-Interacting Protein, UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.

Destabilization of cytochrome c by modified β-cyclodextrin

To clarify the effect of cyclodextrin (CD) on the stability of cytochrome c, the thermal denaturation of cytochrome c was measured by differential scanning calorimetry in aqueous solutions of β-CD modified with three substituents: methyl, acetyl, and 2-hydroxylpropyl groups. The midpoint temperature of thermal denaturation decreased with the addition of modified β-CDs, indicating that cytochrome c was destabilized. The destabilization effect of CD depended on the substituent and increased in the order of acetyl>methyl>2-hydroxypropyl. The estimated binding number and binding constant of the modified β-CDs for cytochrome c are 5.0 ± 1.0 and 10.3 ± 2.9 M−1 for methyl-β-CD, 13.8 ± 3.6 and 4.7 ± 1.6 M−1 for acetyl-β-CD, and 2.8 ± 0.9 and 7.0 ± 3.0 M−1 for 2-hydroxypropyl-β-CD. The destabilization effect of acetyl-β-CD is the highest because many CD molecules interact with proteins by the inclusion effect of CD and the inhibition effect of the acetyl group on the hydrogen bond in the secondary structure. In contrast, the stabilization effect of 2-hydroxypropyl-β-CD is the smallest because the steric exclusion of the 2-hydroxypropyl group inhibits the binding of CD to cytochrome c as compared with the smaller structure of the methyl group. Dependency of the destabilization effect on the molar ratio of CD to cytochrome c suggests that the destabilization effect of CD is caused not only by the “direct” interaction of CD with proteins but also by the “indirect” interaction of CD which promotes the solvation of hydrophobic groups by altering the water structure as observed in urea.

2B1612 シトクロムcの安定性に対する共溶媒効果(水・水和/電解質,第49回日本生物物理学会年会)

2B1612 シトクロムcの安定性に対する共溶媒効果(水・水和/電解質,第49回日本生物物理学会年会)

2P142 cytochrome cの構造安定性に対する水和効果の自由エネルギー解析(水・水和/電解質,第48回日本生物物理学会年会)

2P142 cytochrome cの構造安定性に対する水和効果の自由エネルギー解析(水・水和/電解質,第48回日本生物物理学会年会)

1P111 中温性及び好熱性シトクロームcの熱安定性に及ぼすヘムの効果 : 実験結果と理論解析結果の比較(ヘム蛋白質,第48回日本生物物理学会年会)

1P111 中温性及び好熱性シトクロームcの熱安定性に及ぼすヘムの効果 : 実験結果と理論解析結果の比較(ヘム蛋白質,第48回日本生物物理学会年会)

Role of nuclear-encoded subunit Vb in the assembly and stability of cytochrome c oxidase complex: implications in mitochondrial dysfunction and ROS production

CcO (cytochrome c oxidase) is a multisubunit bigenomic protein complex which catalyses the last step of the mitochondrial electron transport chain. The nuclear-encoded subunits are thought to have roles either in regulation or in the structural stability of the enzyme. Subunit Vb is a peripheral nuclear-encoded subunit of mammalian CcO that is dramatically reduced under hypoxia. Although it has been shown to contain different ligand-binding sites and undergo modifications, its precise function is not known. In the present study we generated a cell line from RAW 264.7 murine macrophages that has a more than 80% reduced level of Vb. Functional analysis of these cells showed a loss of CcO activity, membrane potential and less ability to generate ATP. Resolution of complexes on blue native gel and two-dimensional electrophoretic analysis showed an accumulation of subcomplexes of CcO and also reduced association with supercomplexes of the electron transfer chain. Furthermore, the mitochondria from CcO Vb knock-down cells generated increased ROS (reactive oxygen species), and the cells were unable to grow on galactose-containing medium. Pulse-chase experiments suggest the role of the CcO Vb subunit in the assembly of the complex. We show for the first time the role of a peripheral, non-transmembrane subunit in the formation as well as function of the terminal CcO complex.

Conformational stability of cytochrome b 5, enhanced green fluorescent protein, and their fusion protein Hmwb5-EGFP

The conformational stabilities of chimeric protein Hmwb(5)-EGFP and its constituents (cytochrome b(5) and enhanced green fluorescent protein) in guanidine hydrochloride solutions are reported in this paper. Intensity of fluorescence of tryptophan residues, intensity of EGFP fluorescence in the visible region, absorbance of cytochrome b(5) heme and EGFP fluorophore, and fluorescence anisotropy were used to follow the unfolding process. Thermodynamic parameters of protein unfolding were obtained using different approaches. The data were analyzed using a two-stage model and a linear extrapolation method. Unfolding of protein molecules was additionally monitored by measuring Stern-Volmer constants for tryptophan fluorescence quenching by acrylamide, cesium, and iodide. The accessibility of tryptophan residues of both components in the fusion molecule is lower than in the separate molecules. The thermodynamic stability of the protein globules in the fusion protein is much lower than in the individual protein molecules in solution, the difference in free energy of unfolding being more considerable for cytochrome b(5) (29 +/- 4 and 13 +/- 2 kJ/mol) than for EGFP (26 +/- 0.9 and 20 +/- 2.7 kJ/mol). The data indicate that artificial protein fusion can greatly affect total structural stability, and in the case of cytochrome b(5) and EGFP it results in decrease in free energy of transition from native to denatured unfolded form and consequently to decrease in thermodynamic stability of protein globules compared to the separate proteins.

Solubility and Stability of Cytochrome c in Hydrated Ionic Liquids: Effect of Oxo Acid Residues and Kosmotropicity

Hydrated ionic liquids (ILs) were prepared by adding appropriate amounts of water to hydrophilic ILs. Some hydrated ILs show excellent solubilizing ability for proteins, keeping the basic properties of ILs. The solubility of cytochrome c (cyt c) depended on the structure of the component ions. When component anions have oxo acid residues, the resulting hydrated ILs solubilize cyt c quite well. In such hydrated ILs, the structure and activity of cyt c is influenced by the kosmotropicity of the component ions. We synthesized ILs from various ions having different kosmotropicity, including dihydrogen phosphate (dhp), dibutylphosphate, acetate, lactate, and methanesulfonate as anions. The activity of the dissolved cyt c depends on the permutations of kosmotropicity of the component ions. cyt c shows no structural change and retains its activity when dissolved in the hydrated choline dhp, which is an excellent combination of chaotropic cation and kosmotropic anion. Furthermore, cyt c dissolved in the hydrated choline dhp remained in a native state and was active after 18 months of storage at room temperature.

Insights into the role of the histidines in the structure and stability of cytochrome c

In this paper we investigate the role played by each histidine in the amino acid sequence of yeast iso-1-cytochrome c (with the exception of H18, the residue axially coordinated to the heme iron) in determining the protein structure and stability. To this end, we have generated and characterized the double mutants H26Y/H33Y, H26Y/H39K and H33Y/H39K obtained from the C102T variant of the protein, which retain only one histidine side chain in the amino acid sequence. In particular, the H39K mutation inserts a lysine at position 39 as in the sequence of equine cytochrome c. The H26Y/H33Y/H39K triple mutant, which lacks all three histidines, was also produced and its spectroscopic properties are compared with those of the double mutants. The data highlight the critical role played by H26 in determining protein stability. Recombinant horse cytochrome c and the corresponding H26Y mutant were also generated and characterized. Since equine cytochrome c exhibits higher stability than the yeast protein, this provides a valuable opportunity to understand the role played by the invariant H26 residue in determining structure and stability.

3P157 分子動力学シミュレーションによるシトクロムc_&lt;551&gt;の構造安定性に関する研究(発生・分化))

3P157 分子動力学シミュレーションによるシトクロムc_&lt;551&gt;の構造安定性に関する研究(発生・分化))

Biochemical characteristics of purified beef liver NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase, an obligatory component of the cytochrome P450 dependent monooxygenase system, was purified to electrophoretic homogeneity from beef liver microsomes. The purification procedure involved the ion exchange chromatography of the detergent-solubilized microsomes on first and second DEAE-cellulose columns, followed by 2',5'-ADP Sepharose affinity chromatography. Further concentration of the enzyme and removal of Emulgen 913 and 2'-AMP were accomplished on the final hydroxylapatite column. The enzyme was purified 239-fold and the yield was 13.5%. Monomer molecular weight of the enzyme was estimated to be 76000 +/- 3000 (N = 5) by SDS-PAGE. The absolute absorption spectrum of beef reductase showed two peaks at 455 and 378 nm, with a shoulder at 478 nm, characteristics of flavoproteins. The effects of cytochrome c concentration, pH, and ionic strength on enzyme activity were studied. Reduction of cytochrome c with the enzyme followed Michaelis-Menten kinetics, and the apparent K(m) of the purified enzyme was found to be 47.7 microM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer (pH 7.7). Stability of cytochrome c reductase activity was examined at 25 and 37 degrees C in the presence and absence of 20% glycerol. The presence of glycerol enhanced the stability of cytochrome c reductase activity at both temperatures. Sheep lung microsomal cytochrome P4502B and NADPH-cytochrome P450 reductase were also purified by the already existing methods developed in our laboratory. Both beef liver and sheep lung reductases were found to be effective in supporting benzphetamine and cocaine N-demethylation reactions in the reconstituted systems containing purified sheep lung cytochrome P4502B and synthetic lipid, phosphatidylcholine dilauroyl.

A tale of two charges: Distinct roles for an acidic and a basic amino acid in the structure and function of cytochromec

Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria. As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history. Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp. There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined. Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electro statically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular). This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86. Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90.Key words: cytochrome c, structure-function studies, molecular modelling, surface electrostatics.

A tale of two charges: Distinct roles for an acidic and a basic amino acid in the structure and function of cytochrome &lt;i&gt;c&lt;/i&gt;

Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria. As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history. Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp. There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined. Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electrostatically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular). This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86. Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90.

Effects of salts of alkali earth metals and calcium chloride on the stability of cytochrome c and myoglobin

This study suggests a procedure by which binding of denaturants could be detected without any additional information other than that provided in the denaturation profiles of proteins. Two predominantly α-proteins, namely ferricytochrome c and metmyoglobin were denatured by guanidine hydrochloride (GdnHCl) in the presence of low fixed concentrations of salts at 25°C and transition between native and denatured states was followed by absorbance measurements in the visible region (500-350 nm). The raw data were converted into transition curves from which C m the midpoint of GdnHCl-induced transition, and ΔG app, the free energy changes on denaturation were calculated assuming a two-state mechanism, and values of ΔG app at zero concentration of the denaturant were estimated. It has been observed (1) that chlorides of Na, K, Cs, and Rb do not affect the native conformation of proteins, (2) that GdnHCl-induced denaturations of proteins in presence and absence of sodium bromide, sodium perchlorate and salts of lithium and calcium are reversible, (3) that optical properties of the GdnHCl-denatured state of proteins remain unchanged in presence of the second denaturant, (4) C m, decreases with an increase in the denaturant concentration, and (5) that except for GdnHCl there exist one or more binding sites on the native proteins for the denaturants.

Proton linkage in formation of the cytochrome c-cytochrome c peroxidase complex: electrostatic properties of the high- and low-affinity cytochrome binding sites on the peroxidase.

The electrostatic character of cytochrome c-cytochrome c peroxidase complex formation has been studied by potentiometric titration between pH 5.5 and 7.75. Potentiometric data obtained at ionic strength > or = 100 mM were adequately analyzed in terms of 1:1 complex formation while the simplest model capable of fitting similar data obtained at lower ionic strength involves the assumption of two inequivalent binding sites for the cytochrome on the peroxidase. The stability of cytochrome c binding at the high-affinity site is ca. three orders of magnitude greater than that observed for the low-affinity site and is optimal between pH 6.75 and 7. The electrostatic properties of the two binding sites are distinctly different because, at most values of pH, binding of cytochrome c to the high-affinity site results in proton release while binding of the cytochrome to the low-affinity site results in proton uptake. Furthermore, binding of the cytochrome to the low-affinity site appears to be least stable in the pH range where binding to the high-affinity site is optimal. Interestingly, the binding parameters derived from these measurements were independent of temperature, consistent with a substantial entropic contribution to complex stability. Ferricytochrome c binds to the peroxidase with a slightly greater affinity than does ferrocytochrome c, and no evidence for specific anion effects on complex stability was observed. At low ionic strength (< or = 50 mM) and high pH (7.75), the interaction of the two proteins is more complex and cannot be adequately analyzed in terms of the two-site model.