Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate.

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

Light-Driven Electron Uptake from Nonfermentative Organic Matter to Expedite Nitrogen Dissimilation by Chemolithotrophic Anammox Consortia.

Nonphotosynthetic microorganisms are typically unable to directly utilize light energy, but light might change the metabolic pathway of these bacteria indirectly by forming intermediates such as reactive oxygen species (ROS). This work investigated the role of light on nitrogen conversion by anaerobic ammonium oxidation (anammox) consortia. The results showed that high intensity light (>20000 lx) caused ca. 50% inhibition of anammox activity, and total ROS reached 167% at 60,000 lx. Surprisingly, 200 lx light was found to induce unexpected promotion of the nitrogen conversion rate, and ultraviolet light (<420 nm) was identified as the main contributor. Metagenomic and metatranscriptomic analyses revealed that the gene encoding cytochrome c peroxidase was highly expressed only under 200 lx light. 15N isotope tracing, gene abundance quantification, and external H2O2 addition experiments showed that photoinduced trace H2O2 triggered cytochrome c peroxidase expression to take up electrons from extracellular nonfermentative organics to synthesize NADH and ATP, thereby expediting nitrogen dissimulation of anammox consortia. External supplying reduced humic acid into a low-intensity light exposure system would result in a maximal 1.7-fold increase in the nitrogen conversion rate. These interesting findings may provide insight into the niche differentiation and widespread nature of anammox bacteria in natural ecotopes.

Improved Metabolic Pathways of Glycolysis, Glycogen Synthesis, the Urea Cycle, and Cytochrome Peroxidase Oxidative Reabsorption in a Miniature Bioreactor.

BACKGROUND/AIMS: Bioreactor-based bioartificial liver support systems have had limited success in a translational setting and at preclinical stages. None of the existing systems monitor the metabolic pathways of glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption. Herein, we designed a bioreactor that mimics the human liver microenvironment in vivo and monitors different hepatic metabolic pathways in order to help establish in vitro culture conditions for improved glycolysis, glycogen synthesis, the urea cycle, cytochrome peroxidase oxidative reabsorption and improved hepatic functions in a miniature bioartificial liver. An abnormality in such pathways negatively influences survivability and hepatic functions, including spontaneous liver regeneration. METHODS: We investigated the metabolic functions of primary mouse adult hepatocytes cultured in a three-dimensional configuration under direct oxygenation conditions (5%, 10%, 20%, and 40% O2) for 14 days in the bioreactor. We analyzed the expression of the genes of hepatic metabolic pathways, such as glycolysis (glucokinase, phosphofructokinase, and pyruvate kinase), glycogen synthesis (glycogen synthetase, UTP glucose-1-phosphate uridylylisomerase, phosphoglucomutase, and glycogen phosphorylase), the urea cycle (arginase, ornithine carbomoyltransferase, fumarate hydratase), oxidative reabsorption (peroxidase), and cytochrome peroxides (catalase and superoxide dismutase), and compared it with the level in vivo. The metabolic mini-map was used to represent the above-mentioned metabolic genes. RESULTS: Increased urea secretion under normoxia and hyperoxia conditions (20% and 40% O2, respectively) was observed, while albumin secretion was decreased in hyperoxic cultures. Lactate formation was up to 15 mg/L-g/h-h/106 cells, 2 mg/L-g/h-h/106 cells, and 0.2 mg/L-c/h-h/106 cells in 5%, 20%, and 40% O2 conditions, respectively while glucose consumption was enhanced under hypoxic conditions (5% and 10% O2). Cellular membrane integrity was estimated by lactate dehydrogenase assay and was found to be negligible in only 20% and 40% O2 conditions. The expression of the phase II enzyme UDP-glucuronosyltransferase was only upregulated in 20% oxygenation. CONCLUSION: Taken together, 20% O2 was found to be an optimal condition for the long-term culture (up to 14 days) of hepatocytes that promoted the expression of genes in metabolic pathways such as glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption, and improved hepatic functions in a miniature bioreactor for bioartificial liver construction.

Computational analysis of the tryptophan cation radical energetics in peroxidase Compound I.

Three well-characterized heme peroxidases (cytochrome c peroxidase = CCP, ascorbate peroxidase = APX, and Leishmania major peroxidase = LMP) all have a Trp residue tucked under the heme stacked against the proximal His heme ligand. The reaction of peroxidases with H2O2 to give Compound I results in the oxidation of this Trp to a cationic radical in CCP and LMP but not in APX. Considerable experimental data indicate that the local electrostatic environment controls whether this Trp or the porphyrin is oxidized in Compound I. Attempts have been made to place the differences between these peroxidases on a quantitative basis using computational methods. These efforts have been somewhat limited by the approximations required owing to the computational cost of using fully solvated atomistic models with well-developed forcefields. This now has changed with available GPU computing power and the associated development of software. Here we employ thermodynamic integration and multistate Bennett acceptance ratio methods to help fine-tune our understanding on the energetic differences in Trp radical stabilization in all three peroxidases. These results indicate that the local solvent structure near the redox active Trp plays a significant role in stabilization of the cationic Trp radical.

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed.

Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

The redox potential of a heme cofactor in Nitrosomonas europaea cytochrome c peroxidase: a polarizable QM/MM study.

Redox reactions are crucial to biological processes that protect organisms against oxidative stress. Metalloenzymes, such as peroxidases which reduce excess reactive oxygen species into water, play a key role in detoxification mechanisms. Here we present the results of a polarizable QM/MM study of the reduction potential of the electron transfer heme in the cytochrome c peroxidase of Nitrosomonas europaea. We have found that environment polarization does not substantially affect the computed value of the redox potential. Particular attention has been given to analyzing the role of electrostatic interactions within the protein environment and the solvent on tuning the redox potential of the heme co-factor. We have found that the electrostatic interactions predominantly explain the fluctuations of the vertical ionization/attachment energies of the heme for the sampled configurations, and that the long range electrostatic interactions (up to 40 Å) contribute substantially to the absolute values of the vertical energy gaps.

A Binuclear CuA Center Designed in an All α-Helical Protein Scaffold.

The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein coregulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here, we present a fully functional CuA center designed in a structurally nonhomologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that CuACcP is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from extended X-ray absorption fine structure spectroscopy and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu(II) and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties in a metal redox center.

A Stable Ferryl Porphyrin at the Active Site of Y463M BthA.

BthA is a diheme enzyme that is a member of the bacterial cytochrome c peroxidase superfamily, capable of generating a highly unusual Fe(IV)Fe(IV)═O oxidation state, known to be responsible for long-range oxidative chemistry in the enzyme MauG. Here, we show that installing a canonical Met ligand in lieu of the Tyr found at the heme of MauG associated with electron transfer, results in a construct that yields an unusually stable Fe(IV)═O porphyrin at the peroxidatic heme. This state is spontaneously formed at ambient conditions using either molecular O2 or H2O2. The resulting data illustrate how a ferryl iron, with unforeseen stability, may be achieved in biology.

The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy.

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant KD of 20±4×10-6  M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.

Structure elaboration of isoniazid: synthesis, in silico molecular docking and antimycobacterial activity of isoniazid-pyrimidine conjugates.

Designing small molecule-based new drug candidates through structure modulation of the existing drugs has drawn considerable attention in view of inevitable emergence of resistance. A new series of isoniazid-pyrimidine conjugates were synthesized in good yields and evaluated for antitubercular activity against the H37Rv strain of Mycobacterium tuberculosis using the microplate Alamar Blue assay. Structure-anti-TB relationship profile revealed that conjugates 8a and 8c bearing a phenyl group at C-6 of pyrimidine scaffold were most active (MIC99 10 µM) and least cytotoxic members of the series. In silico docking of 8a in the active site of bovine lactoperoxidase as well as a cytochrome C peroxidase mutant N184R Y36A revealed favorable interactions similar to the heme enzyme catalase peroxidase (KatG) that activates isoniazid. This investigation suggests a rationale for further work on this promising series of antitubercular agents.

Escherichia coli cytochrome c peroxidase is a respiratory oxidase that enables the use of hydrogen peroxide as a terminal electron acceptor.

Microbial cytochrome c peroxidases (Ccp) have been studied for 75 years, but their physiological roles are unclear. Ccps are located in the periplasms of bacteria and the mitochondrial intermembrane spaces of fungi. In this study, Ccp is demonstrated to be a significant degrader of hydrogen peroxide in anoxic Escherichia coli Intriguingly, ccp transcription requires both the presence of H2O2 and the absence of O2 Experiments show that Ccp lacks enough activity to shield the cytoplasm from exogenous H2O2 However, it receives electrons from the quinone pool, and its flux rate approximates flow to other anaerobic electron acceptors. Indeed, Ccp enabled E. coli to grow on a nonfermentable carbon source when H2O2 was supplied. Salmonella behaved similarly. This role rationalizes ccp repression in oxic environments. We speculate that micromolar H2O2 is created both biologically and abiotically at natural oxic/anoxic interfaces. The OxyR response appears to exploit this H2O2 as a terminal oxidant while simultaneously defending the cell against its toxicity.

How Does Replacement of the Axial Histidine Ligand in Cytochrome c Peroxidase by Nδ-Methyl Histidine Affect Its Properties and Functions? A Computational Study.

Heme peroxidases have important functions in nature related to the detoxification of H2O2. They generally undergo a catalytic cycle where, in the first stage, the iron(III)-heme-H2O2 complex is converted into an iron(IV)-oxo-heme cation radical species called Compound I. Cytochrome c peroxidase Compound I has a unique electronic configuration among heme enzymes where a metal-based biradical is coupled to a protein radical on a nearby Trp residue. Recent work using the engineered Nδ-methyl histidine-ligated cytochrome c peroxidase highlighted changes in spectroscopic and catalytic properties upon axial ligand substitution. To understand the axial ligand effect on structure and reactivity of peroxidases and their axially Nδ-methyl histidine engineered forms, we did a computational study. We created active site cluster models of various sizes as mimics of horseradish peroxidase and cytochrome c peroxidase Compound I. Subsequently, we performed density functional theory studies on the structure and reactivity of these complexes with a model substrate (styrene). Thus, the work shows that the Nδ-methyl histidine group has little effect on the electronic configuration and structure of Compound I and little changes in bond lengths and the same orbital occupation is obtained. However, the Nδ-methyl histidine modification impacts electron transfer processes due to a change in the reduction potential and thereby influences reactivity patterns for oxygen atom transfer. As such, the substitution of the axial histidine by Nδ-methyl histidine in peroxidases slows down oxygen atom transfer to substrates and makes Compound I a weaker oxidant. These studies are in line with experimental work on Nδ-methyl histidine-ligated cytochrome c peroxidases and highlight how the hydrogen bonding network in the second coordination sphere has a major impact on the function and properties of the enzyme.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome†c Peroxidase with an Additional, Distant Electrostatic Binding Site.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.

Dual species transcript profiling during the interaction between banana (Musa acuminata) and the fungal pathogen Fusarium oxysporum f. sp. cubense.

BACKGROUND: Banana wilt disease, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), is one of the most devastating diseases in banana (Musa spp.). Foc is a soil borne pathogen that causes rot of the roots or wilt of leaves by colonizing the xylem vessels. The dual RNA sequencing is used to simultaneously assess the transcriptomes of pathogen and host. This method greatly helps to understand the responses of pathogen and host to each other and discover the potential pathogenic mechanism. RESULTS: Plantlets of two economically important banana cultivars, Foc TR4 less susceptible cultivar NK and susceptible cultivar BX, were used to research the Foc-banana interaction mechanism. Notably, the infected NK had more significantly up-regulated genes on the respiration machinery including TCA cycle, glyoxylate, glycerol, and glycolysis compared to BX at 27 h post inoculation (hpi). In addition, genes involved in plant-pathogen interaction, starch, sucrose, linolenic acid and sphingolipid metabolisms were uniquely more greatly induced in BX than those in NK during the whole infection. Genes related to the biosynthesis and metabolism of SA and JA were greatly induced in the infected NK; while auxin and abscisic acid metabolisms related genes were strongly stimulated in the infected BX at 27 hpi. Furthermore, most of fungal genes were more highly expressed in the roots of BX than in those of NK. The fungal genes related to pathogenicity, pectin and chitin metabolism, reactive oxygen scavenging played the important roles during the infection of Foc. CCP1 (cytochrome c peroxidase 1) was verified to involve in cellulose utilization, oxidative stress response and pathogenicity of fungus. CONCLUSION: The transcriptome indicated that NK had much faster defense response against Foc TR4 than BX and the expression levels of fungal genes were higher in BX than those in NK. The metabolisms of carbon, nitrogen, and signal transduction molecular were differentially involved in pathogen infection in BX and NK. Additionally, the putative virulence associated fungal genes involved in colonization, nutrition acquirement and transport provided more insights into the infection process of Foc TR4 in banana roots.

Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping.

Transient tyrosine and tryptophan radicals play key roles in the electron transfer (ET) reactions of photosystem (PS) II, ribonucleotide reductase (RNR), photolyase, and many other proteins. However, Tyr and Trp are not functionally interchangeable, and the factors controlling their reactivity are often unclear. Cytochrome c peroxidase (CcP) employs a Trp191•+ radical to oxidize reduced cytochrome c (Cc). Although a Tyr191 replacement also forms a stable radical, it does not support rapid ET from Cc. Here we probe the redox properties of CcP Y191 by non-natural amino acid substitution, altering the ET driving force and manipulating the protic environment of Y191. Higher potential fluorotyrosine residues increase ET rates marginally, but only addition of a hydrogen bond donor to Tyr191• (via Leu232His or Glu) substantially alters activity by increasing the ET rate by nearly 30-fold. ESR and ESEEM spectroscopies, crystallography, and pH-dependent ET kinetics provide strong evidence for hydrogen bond formation to Y191• by His232/Glu232. Rate measurements and rapid freeze quench ESR spectroscopy further reveal differences in radical propagation and Cc oxidation that support an increased Y191• formal potential of ∼200 mV in the presence of E232. Hence, Y191 inactivity results from a potential drop owing to Y191•+ deprotonation. Incorporation of a well-positioned base to accept and donate back a hydrogen bond upshifts the Tyr• potential into a range where it can effectively oxidize Cc. These findings have implications for the YZ/YD radicals of PS II, hole-hopping in RNR and cryptochrome, and engineering proteins for long-range ET reactions.

Resonance Raman, Electron Paramagnetic Resonance, and Magnetic Circular Dichroism Spectroscopic Investigation of Diheme Cytochrome c Peroxidases from Nitrosomonas europaea and Shewanella oneidensis.

Cytochrome c peroxidases (bCcPs) are diheme enzymes required for the reduction of H2O2 to water in bacteria. There are two classes of bCcPs: one is active in the diferric form (constitutively active), and the other requires the reduction of the high-potential heme (H-heme) before catalysis commences (reductively activated) at the low-potential heme (L-heme). To improve our understanding of the mechanisms and heme electronic structures of these different bCcPs, a constitutively active bCcP from Nitrosomonas europaea ( NeCcP) and a reductively activated bCcP from Shewanella oneidensis ( SoCcP) were characterized in both the diferric and semireduced states by electron paramagnetic resonance (EPR), resonance Raman (rRaman), and magnetic circular dichroism (MCD) spectroscopy. In contrast to some previous crystallographic studies, EPR and rRaman spectra do not indicate the presence of significant amounts of a five-coordinate, high-spin ferric heme in NeCcP or SoCcP in either the diferric or semireduced state in solution. This observation points toward a mechanism of activation in which the active site L-heme is not in a static, five-coordinate state but where the activation is more subtle and likely involves formation of a six-coordinate hydroxo complex, which could then react with hydrogen peroxide in an acid-base-type reaction to create Compound 0, the ferric hydroperoxo complex. This mechanism lies in stark contrast to the diheme enzyme MauG that exhibits a static, five-coordinate open heme site at the peroxidatic heme and that forms a more stable FeIV═O intermediate.

YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity.

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50 kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be -170 mV, +133 mV and +210 mV, respectively, at pH 7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH 7.0 and millimolar range KM values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (KM = 0.6 ±â€¯0.2 and 1.8 ±â€¯0.5 mM H2O2, respectively), with similar turnover numbers (kcat = 19 ±â€¯2 and 13 ±â€¯2 s-1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV) = O species was observed upon incubation of fully oxidized YhjA with H2O2. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.

LC-MS/MS Proteoform Profiling Exposes Cytochrome c Peroxidase Self-Oxidation in Mitochondria and Functionally Important Hole Hopping from Its Heme.

LC-MS/MS profiling reveals that the proteoforms of cytochrome c peroxidase (Ccp1) isolated from respiring yeast mitochondria are oxidized at numerous Met, Trp, and Tyr residues. In vitro oxidation of recombinant Ccp1 by H2O2 in the absence of its reducing substrate, ferrocytochrome c, gives rise to similar proteoforms, indicating uncoupling of Ccp1 oxidation and reduction in mitochondria. The oxidative modifications found in the Ccp1 proteoforms are consistent with radical transfer (hole hopping) from the heme along several chains of redox-active residues (Trp, Met, Tyr). These modifications delineate likely hole-hopping pathways to novel substrate-binding sites. Moreover, a decrease in recombinant Ccp1 oxidation by H2O2 in vitro in the presence of glutathione supports a protective role for hole hopping to this antioxidant. Isolation and characterization of extramitochondrial Ccp1 proteoforms reveals that hole hopping from the heme in these proteoforms results in selective oxidation of the proximal heme ligand (H175) and heme labilization. Previously, we demonstrated that this labilized heme is recruited for catalase maturation (Kathiresan, M.; Martins, D.; English, A. M. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17468-17473; DOI: 10.1073/pnas.1409692111 ). Following heme release, apoCcp1 exits mitochondria, yielding the extramitochondrial proteoforms that we characterize here. The targeting of Ccp1 for selective H175 oxidation may be linked to the phosphorylation status of Y153 close to the heme since pY153 is abundant in certain proteoforms. In sum, when insufficient electrons from ferrocytochrome c are available to Ccp1 in mitochondria, hole hopping from its heme expands its physiological functions. Specifically, we observe an unprecedented hole-hopping sequence for heme labilization and identify hole-hopping pathways from the heme to novel substrates and to glutathione at Ccp1's surface. Furthermore, our results underscore the power of proteoform profiling by LC-MS/MS in exploring the cellular roles of oxidoreductases.

Striatal mitochondria response to 3-nitropropionic acid and fish oil treatment.

BACKGROUND: Mitochondrial dysfunction is involved in neurodegenerative diseases, such as Huntington's disease (HD). 3-Nitropropionic acid (3-NP) is a mitochondrial toxin that specifically inhibits complex II of the electron transport chain (ETC) and is used to generate an experimental model of HD. OBJECTIVE: To study the effect of fish liver oil (FO) over the mitochondrial dysfunction induced via partial ETC inhibition by 3-NP. METHODS: This study was performed in rats and consisted of two phases: (i) administration of increasing doses of 3-NP and (ii) administration of FO for 14 days before to 3-NP. The rats' exploratory activity; complex I, II, III, and IV activities; and rearing behavior were observed. Additionally, the number of TUNEL-positive cells and various mitochondrial parameters, including oxygen consumption, transmembrane potential, adenosine triphosphate synthesis, and ETC activity, were measured. RESULTS: We observed that FO exerted a protective effect against the 3-NP-induced toxicity, although complex II inhibition still occurred. Instead, this effect was related to strengthened mitochondrial complex III and IV activities. DISCUSSION: Our results show that FO exerts a beneficial prophylactic effect against mitochondrial damage. Elucidating the mechanisms linking the effects of FO with its prevention of neurodegeneration could be the key to developing recommendations for FO consumption in neurological pathologies.

Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins.

Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.