Diminished degradation of yeast cytochrome c by interactions with its physiological partners.

The level and structure of yeast iso-1-cytochrome c and iso-2-cytochrome c, encoded by the nuclear genes CYC1 and CYC7, respectively, are normally not altered in rho- mutants, which completely lack the cytochromes a.a3 subunits and cytochrome b that are encoded by mitochondrial DNA. In contrast, iso-cytochromes c containing the amino acid change Thr-78-->Ile (T78I) were observed at the normal or near-normal wild-type level in rho+ strains but were completely absent in rho- mutants. We have demonstrated with the "global" suppressor mutation Asn-52-->Ile and by pulse-chase labeling that the T78I iso-1-cytochrome c undergoes rapid cellular degradation in rho- mutants. Furthermore, specific mutations revealed that the deficiency of T78I iso-1 cytochrome c can be caused by the lack of cytochrome a.a3 or cytochrome c1, but not by the lack of cytochrome b. Thus, this and certain other, but not all, labile forms of cytochrome c are protected from degradation by the interaction with its physiological partners.

Gas-phase folding and unfolding of cytochrome c cations.

Water is thought to play a dominant role in protein folding, yet gaseous multiply protonated proteins from which the water has been completely removed show hydrogen/deuterium (H/D) exchange behavior similar to that used to identify conformations in solution. Indicative of the gas-phase accessibility to D2O, multiply-charged (6+ to 17+) cytochrome c cations exchange at six (or more) distinct levels of 64 to 173 out of 198 exchangeable H atoms, with the 132 H level found at charge values 8+ to 17+. Infrared laser heating and fast collisions can apparently induce ions to unfold to exchange at a higher distinct level, while charge-stripping ions to lower charge values yields apparent folding as well as unfolding.

Modulation of the Silica Sol–Gel Composition for the Promotion of Direct Electron Transfer to Encapsulated Cytochrome c

The direct electron transfer between indium–tin oxide electrodes (ITO) and cytochrome c encapsulated in different sol–gel silica networks was studied. Cyt c@silica modified electrodes were synthesized by a two-step encapsulation method mixing a phosphate buffer solution with dissolved cytochrome c and a silica sol prepared by the alcohol-free sol–gel route. These modified electrodes were characterized by cyclic voltammetry, UV–vis spectroscopy, and in situ UV–vis spectroelectrochemistry. The electrochemical response of encapsulated protein is influenced by the terminal groups of the silica pores. Cyt c does not present electrochemical response in conventional silica (hydroxyl terminated) or phenyl terminated silica. Direct electron transfer to encapsulated cytochrome c and ITO electrodes only takes place when the protein is encapsulated in methyl modified silica networks.

Cytochrome c(551) from Starkeya novella - Characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulficlophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: + 133 mV (pH 6.0); + 104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxY, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

The acidic nature of the CcmG redox-active center is important for cytochrome c maturation in Escherichia coli

Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes (ccmABC DEFGH). One of these genes, ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.

Nuclear translocation of cytochrome c during apoptosis

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunofluorescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytochrome c in inducing nuclear apoptosis.

Nano-grids of Yeast Cytochrome C

One innovative thought in biomolecular electronics is the exploitation of electron transfer proteins. Using nature's self assembly techniques, proteins can build highly organized edifices with retained functional activity, and they can serve as platforms for biosensors. In this research work, Yeast Cytochrome C (YCC) is immobilized with a help of a linker molecule, 3-Mercaptopropyltrimethoxysilane (3-MPTS) on a hydroxylated surface of a silicon substrate. Atomic Force Microscopy (AFM) is used for characterization. AFM data shows immobilization of one YCC molecule in between eight grids that are formed by the linker molecules. 3-MPTS monolayers are organized in grids that are 1.2 nm apart. Immobilization of 3-MPTS was optimized using a concentration of 5 mM in a completely dehydrated state for 30 minutes. The functionally active grids of YCC can now be incorporated with Cytochrome C oxidase on a Platinum electrode surface for transfer of electrons in development of biosensors, such as nitrate sensor, that are small in size, cheaper, and easier to manufacture than the top-down approach of fabrication of molecular biodevices

Enhancement of the Cytotoxic Effect of Anticancer Agent by Cytochrome c Functionalised Hybrid Nanoparticles in Hepatocellular Cancer Cells

Treatment of hepatocellular cancer with chemotherapeutic agents has limited successin clinical practice and their efficient IC50 concentration would require extremely highdoses of drug administration which could not be tolerated due to systemic side effects.In order to potentiate the efficacy of anticancer agents we explored the potentialof co-treatment with pro-apoptotic Cytochrome c which activates the apoptoticpathway downstream of p53 that is frequently mutated in cancer. To this end weused hybrid iron oxide-gold nanoparticles as a drug delivery system to facilitate theinternalisation of Cytochrome c into cultured HepG2 hepatocellular carcinoma cells.Our results showed that Cytochrome c can be easily conjugated to the gold shell ofthe nanoparticles which are readily taken up by the cells. We used Cytochrome cin concentration (0.2μgmL-1) below the threshold required to induce apoptosis onits own. When the conjugate was administered to cells treated by doxorubicin, itsignificantly reduced its IC50 concentration from 9μgmL-1 to 3.5μgmL-1 as detectedby cell viability assay, and the efficiency of doxorubicin on decreasing viability ofHepG2 cells was significantly enhanced in the lower concentration range between0.01μgmL-1 to 5μgmL-1. The results demonstrate the potential of the application oftherapeutic proteins in activating the apoptotic pathway to complement conventionalchemotherapy to increase its efficacy. The application of hybrid iron oxide-goldnanoparticles can also augment the specificity of drug targeting and could serve as amodel drug delivery system for pro-apoptotic protein targeting and delivery.

Electrochemistry of Microperoxidase-11 (MP-11) on Glassy Carbon Electrode Modified with Chitosan

In this paper, microperoxidase-11 (MP-11) was immobilized on glassy carbon electrode surface modified with chitosan by physical adsorption. The direct electrochemistry and the electrocatalytic behaviours to O-2 and the H2O2 of MP-11 on glassy carbon electrode modified with chitosan were characterized by cyclic voltammetry. The results indicate that MP-11 on modified electrode displays a quasi-reversible electrochemical process coupled with proton transfer in the phosphate buffer solutions(pH = 7.12). Direct electrochemical reaction of MP-11 on modified electrode has been realized. MP-11 on modified electrode can catalyze reduction for O-2 and H2O2. Both of the catalytic reductions are surface-controlled electrochemical process.

Effect of La3+ on structure and electrochemical reaction of microperoxidase-11 in imitated physiological solution

In this paper, the interaction mechanism between La3+ and microperoxidase-11 (MP-11) in the imitated physiological solution was investigated with the electrochemical and spectroscopic methods. It was found that when the molar ratio of La3+, and MP-11 is low, such as 2, La3+ can coordinate with oxygen in the propionic acid group of the heme group in the MP-11 molecule, forming the La-MP-11 complexes and leading to the increase in the non-planarity of the porphyrin cycle in the heme group and then the increase in the extent of exposure of the electrochemically active center, Fe(I I I) in the porphyrin cycle of the heme group. The increase in the extent of exposure of the electrochemically active center, Fe(III) in the porphyrin cycle of the heme group would increase the reversibility of the electrochemical reaction of the La-MP-11 complexes and its electrocatalytic activity for the reduction of H2O2. The results of the chromatographic analysis demonstrated that the average molar ratio of La3+ and MP-11 in the La-MP-11 complexes is 1.62.When the molar ratio of La3+ and MP-11 is high, such as 3, La3+ would shear some amino acid residues of the peptide of MP-11. Therefore, many La3+ ions can bind to the oxygen- and/or nitrogen-containing groups in the sheared amino acid residues except coordinating with the sheared and non-sheared MP-11 molecules.

Hydrogen peroxide biosensor based on microperoxidase-11 entrapped in lipid membrane

A highly catalytic activity microperoxidase-11 (MP-11) biosensor for H2O2 was developed to immobilizing the heme peptide in didodecyldimethylammonium bromide (DDAB) lipid membrane. The enzyme electrode thus obtained responded to H2O2 without electron mediator or promoter, at a potential of +0.10 V versus Ag \ AgCl. A linear calibration curve is obtained over the range from 2.0 x 10(-5) to 2.4 x 10(-3) M. The biosensor responds to hydrogen peroxide in 15 s and has a detection limit of 8 x 10(-7) M (S/N = 3) Providing a natural environment with lipid membrane for protein immobilization and maintenance of protein functions is a suitable option for the design of biosensors.

Promoter effect of La3+ on the electrochemical reaction of microperoxidase-11

The effect of La3+ on the electrochemical behavior and structure of heme undecapeptide-microperoxidase-11 (MP-11)-in the aqueous solution was investigated using cyclic voltammetry, circular dichroism (CD) and UV-vis absorption spectrometry. It was found for the first time that La3+ would promote the electrochemical reaction of MP-11 at the glassy carbon (GC) electrode. This is mainly due to the fact that La3+ would induce more beta-turn and alpha-helical conformations from the random coil conformation of MP-11 and increase the non-planarity of the heme.

Surface-enhanced Raman spectroscopy of microperoxidase-11 on roughed silver electrodes

The conformation of microperoxidase-11 (MP-11) adsorbed on roughened silver electrodes was studied using surface-enhanced Fourier transform Raman spectroscopy. The results demonstrate that MP-11 was initially adsorbed via its polypeptide chain with a alpha-helix conformation, as indicated by the enhancement of the characteristic bands related to the amides I and III. The weak resonance effect of the porphyrin macrocycle in the near IR region contributes to the spectrum of the heme group. The presence of imidazole as the sixth ligand to the heme iron influences the conformation of the polypeptide chain of MP-11 on the electrode surface. Evaporation of solvent water results in an opened conformation of the adsorbed MP-11. which allows the heme group to contact the electrode surface directly.

Resonance Raman and surface-enhanced Raman spectroscopic study of microperoxidase-11

The electron transfer and structure of microperoxidase-11(MP-11) in solution and at electrode/solution interface were studied by electrochemical, resonance Raman and surface-enhanced Raman spectroscopic techniques. Results show that the central iron in heme group was six-coordinated in solution, whereas it was converted to five-coordinated state as MP-11 was adsorbed on the surface of a roughened silver electrode, due to the reorientation of MP-11 molecules. The electrochemical properties of MP-11 were directly affected by the coordination state of heme iron.

Electrochemistry and spectroscopy study on the interaction of microperoxidase-11 with lipid membrane

The interaction of microperoxidase-11 (MP11) with cationic lipid vesicles of didodecyldimethylammonium bromide (DDAB) induces an alpha -helical conformation from random coil conformations in solution and this change then makes heme macrocycle more distorted. DDAB-induced MP11 conformations were investigated by cyclic votammetry (CV), circular dichroism (CD) and UV-vis spectrometry. All results indicate that the binding of MP11 in solution to DDAB vesicles and the ordered structure formation are driven by mostly electrostatic interaction between negatively charged residues in the undecapeptide and positively charged lipid headgroups on the membrane surface. Upon binding to DDAB, its half-peak potential was also changed. The mechanism of the interaction between MP11 and DDAB was also discussed. (C) 2001 Elsevier Science B.V. All rights reserved.