Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.     

Cytochrome c methylation

In this review, protein methylation is outlined in general terms, highlighting the major amino acids that are methylated and some of the proteins in which they are found. The majority of the review examines the methylation of cytochrome c at Lys-77 of lower eukaryotes as a possible model for methylation studies. Early work involving the purification and characterization of the methyltransferase responsible for this methylation indicated cytochrome c was methylated posttranslationally, yet prior to import into the mitochondria. Methylation in vitro occurred only at the in vivo methylation site and only on cytochrome c. Later studies using in vitro translated apocytochrome c revealed that methylated, as compared with unmethylated, apocytochrome c was imported preferentially into yeast, but not rat liver, mitochondria. Efforts to discover the reasons for this preference have shown that methylation of apocytochrome c dramatically lowers its isoelectric point (against a predicted increase) and decrease its Stokes radius. A possible mechanism for these differences involving the disruption of hydrogen bonds is presented here with space-filling models. Finally, the in vivo significance of this modification is also discussed.     

Cytochrome c: functions beyond respiration

Cytochrome c is primarily known for its function in the mitochondria as a key participant in the life-supporting function of ATP synthesis. However, when a cell receives an apoptotic stimulus, cytochrome c is released into the cytosol and triggers programmed cell death through apoptosis. The release of cytochrome c and cytochrome-c-mediated apoptosis are controlled by multiple layers of regulation, the most prominent players being members of the B-cell lymphoma protein-2 (BCL2) family. As well as its role in canonical intrinsic apoptosis, cytochrome c amplifies signals that are generated by other apoptotic pathways and participates in certain non-apoptotic functions.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

State of heme in heme c systems: Cytochrome c and heme c models

Polarized resonance Raman spectra of horse heart ferricytochrome c as a function of pH in the range 1.0–12, in the presence of the extrinsic ligands imidazole, cyanide, and azide, and in 4 M urea, are reported, as are resonance Raman spectra of heme undecapeptide in the presence of imidazole, pH 6.8 and pH 2.0, and with cyanide at pH 6.8. The range of investigation is 140–1700 cm^?1, using the 5145-, 4880-, and 4579-Å excitations. The spectra have been analyzed in terms of complexity, sensitivity, and the conformation-heme energetics of the systems. The state of heme in various forms is analyzed with regard to heme energetics, core size, nature of planarity, and coordination configuration. All low-spin forms of heme c systems, cytochrome c, and heme models are concluded to be hexacoordinated, in-plane heme iron systems. The effect of the location of the heme in the protein environment is found to be a slight expansion of the porphyrin core, ～0.01 Å, while the covalent linkage of heme to protein and a mixed nature of axial coordination configuration seem to have little effect on the energetics of the heme group. Complex formation with extrinsic ligand, imidazole, cyanide, or azide, results in a slight contraction of the heme core. The formation of cytochrome c form IV, the alkaline form, is shown to follow a process with apK _a of about 8.4, and similarly, acidic form II is created following the prior formation of an intermediate form with apK _a of about 3.6. The precursor to form IV is interpreted as containing perturbation of the pyrrol rings, whereas the precursor to the acidic form seems to reflect alteration of the energetics of the C_αC_{m α} structures of the heme group. The acidic form of heme undecapeptide is a hexacoordinated high-spin heme with an estimated displacement of 0.25 Å from the heme plane. The pH 2 form of cytochrome c is also a hexacoordinated high-spin form with two weak axial ligands, but iron is in the plane of the porphyrin ring.     

Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.     

Cytochrome c peroxidase from a methylotrophic yeast: physiological role and isolation

Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth⁺) in the absence of catalase activity. These Mth⁺ gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type strain. In contrast to the parental C-105 strain, H₂O₂ does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor strains is strongly correlated with increased levels of the alternative H₂O₂-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized. Received: 9 December 1996 / Received revision: 5 May 1997 / Accepted: 25 May 1997     

Cytochrome c folding dynamics

Understanding how denatured polypeptides self-assemble into correctly folded native protein structures is one of the grand challenges of 21st century science. Cytochrome c refolding has been exhaustively studied with a vast array of triggers and spectroscopic probes. Within the past year, atomically detailed dynamics simulations of this process have appeared as well. This wealth of data provides insights into the conformations and dynamics of diverse parts of the polypeptide at many stages in the refolding reaction. The combination of innovative experiments and powerful computational methods promises to produce a model that reconciles many disparate observations of cytochrome c folding.     

线粒体、细胞色素c与细胞凋亡

线粒体是细胞的一个独特而重要的细胞器,它为细胞各种生命活动提供能量。许多研究表明,线粒体的作用远比人们了解的复杂和多样。近年来研究发现,线粒体与细胞凋亡密切相关,表现在如下一些方面。１．细胞凋亡早期线粒体跨膜电位（ΔΨｍ）下降自１９９３年以来,Ｋｒｏ...     

Cytochrome c and insect cell apoptosis

Abstract The role of cytochrome c in insect cell apoptosis has drawn considerable attention and has been subject to considerable controversy. In Drosophila, the majority of studies have demonstrated that cytochrome c may not be involved in apoptosis, although there are conflicting reports. Cytochrome c is not released from mitochondria into the cytosol and activation of the initiator caspase Dronc or effector caspase Drice is not associated with cytochrome c during apoptosis in Drosophila SL2 cells or BG2 cells. Cytochrome c failed to induce caspase activation and promote caspase activation in Drosophila cell lysates, but remarkably caused caspase activation in extracts from human cells. Knockdown of cytochrome c does not protect cells from apoptosis and over‐expression of cytochrome c also does not promote apoptosis. Structural analysis has revealed that cytochrome c is not required for Dapaf‐1 complex assembly. In Lepidoptera, the involvement of cytochrome c in apoptosis has been demonstrated by the accumulating evidence. Cytochrome c release from mitochondria into cytosol has been observed in different cell lines such as Spodoptera frugiperda Sf9, Spodoptera litura Sl‐1 and Lymantria dispar LdFB. Silencing of cytochrome c expression significantly affected apoptosis and activation of caspase and the addition of cytochrome c to cell‐free extracts results in caspase activation, suggesting the activation of caspase is dependent on cytochrome c. Although Apaf‐1 has not been identified in Lepidoptera, the inhibitor of apoptosome formation can inhibit apoptosis and caspase activation. Cytochrome c may be exclusively required for Lepidoptera apoptosis.