N-acetylcysteine and cysteamine bitartrate prevent azide-induced neuromuscular decompensation by restoring glutathione balance in two novel surf1-/- zebrafish deletion models of Leigh syndrome.

SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression and progressive multisystem dysfunction. Here, we describe two novel surf1-/- zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1-/- mutants manifested adult-onset ocular anomalies and decreased swimming activity, as well as classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate. surf1-/- larvae also demonstrated oxidative stress and stressor hypersensitivity to the complex IV inhibitor, azide, which exacerbated their complex IV deficiency, reduced supercomplex formation, and induced acute neurodegeneration typical of LS including brain death, impaired neuromuscular responses, reduced swimming activity, and absent heartrate. Remarkably, prophylactic treatment of surf1-/- larvae with either cysteamine bitartrate or N-acetylcysteine, but not other antioxidants, significantly improved animal resiliency to stressor-induced brain death, swimming and neuromuscular dysfunction, and loss of heartbeat. Mechanistic analyses demonstrated cysteamine bitartrate pretreatment did not improve complex IV deficiency, ATP deficiency, or increased tissue lactate but did reduce oxidative stress and restore glutathione balance in surf1-/- animals. Overall, two novel surf1-/- zebrafish models recapitulate the gross neurodegenerative and biochemical hallmarks of LS, including azide stressor hypersensitivity that was associated with glutathione deficiency and ameliorated by cysteamine bitartrate or N-acetylcysteine therapy.

Dichloroacetate and thiamine improve survival and mitochondrial stress in a C. elegans model of dihydrolipoamide dehydrogenase deficiency.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disorder caused by depletion of DLD from α-ketoacid dehydrogenase complexes. Caenorhabditis elegans animal models of DLD deficiency generated by graded feeding of dld-1(RNAi) revealed that full or partial reduction of DLD-1 expression recapitulated increased pyruvate levels typical of pyruvate dehydrogenase complex deficiency and significantly altered animal survival and health, with reductions in brood size, adult length, and neuromuscular function. DLD-1 deficiency dramatically increased mitochondrial unfolded protein stress response induction and adaptive mitochondrial proliferation. While ATP levels were reduced, respiratory chain enzyme activities and in vivo mitochondrial membrane potential were not significantly altered. DLD-1 depletion directly correlated with the induction of mitochondrial stress and impairment of worm growth and neuromuscular function. The safety and efficacy of dichloroacetate, thiamine, riboflavin, 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), l-carnitine, and lipoic acid supplemental therapies empirically used for human DLD disease were objectively evaluated by life span and mitochondrial stress response studies. Only dichloroacetate and thiamine showed individual and synergistic therapeutic benefits. Collectively, these C. elegans dld-1(RNAi) animal model studies demonstrate the translational relevance of preclinical modeling of disease mechanisms and therapeutic candidates. Results suggest that clinical trials are warranted to evaluate the safety and efficacy of dichloroacetate and thiamine in human DLD disease.

Kinetic Isotope Effect Analysis of RNA 2'-O-Transphosphorylation.

The breaking of RNA strands by 2'-O-transphosphorylation is a ubiquitous reaction in biology, and enzymes that catalyze this reaction play key roles in RNA metabolism. The mechanisms of 2'-O-transphosphorylation in solution are relatively well studied, but complex and can involve different transition states depending on how the reaction is catalyzed. Because of this complexity and the lack of experimental information on transition-state structure, pinning down the chemical details of enzyme-catalyzed RNA strand cleavage has been difficult. Kinetic isotope effects (KIEs) provide information about changes in bonding as a reaction proceeds from ground state to transition state, and therefore they provide a powerful tool for revealing mechanistic detail. Application of kinetic isotope analyses to RNA 2'-O-transphosphorylation faces three fundamental challenges: synthesis of RNA substrate isotopomers with 18O substitutions at the 2'-O, 5'-O and nonbridging phosphoryl oxygens; determination of the 18O/16O ratios in the residual unreacted substrate or product RNAs; and analyzing these data to allow calculation of the KIEs for use in evaluating different mechanistic scenarios. In this chapter, we outline methods for surmounting these challenges for solution RNA 2'-O-transphosphorylation reactions, and we describe their initial application to understand nonenzymatic solution reactions and reactions catalyzed by the enzyme ribonuclease A.

Isotope effect analyses provide evidence for an altered transition state for RNA 2'-O-transphosphorylation catalyzed by Zn(2+).

Solvent D2O and (18)O kinetic isotope effects on RNA 2'-O-transphosphorylation catalyzed by Zn(2+) demonstrate an altered transition state relative to specific base catalysis. A recent model from DFT calculations involving inner sphere coordination to the non-bridging and leaving group oxygens is consistent with the data.

Multiple alternative substrate kinetics.

The specificity of enzymes for their respective substrates has been a focal point of enzyme kinetics since the initial characterization of metabolic chemistry. Various processes to quantify an enzyme's specificity using kinetics have been utilized over the decades. Fersht's definition of the ratio kcat/Km for two different substrates as the "specificity constant" (ref [7]), based on the premise that the important specificity existed when the substrates were competing in the same reaction, has become a consensus standard for enzymes obeying Michaelis-Menten kinetics. The expansion of the theory for the determination of the relative specificity constants for a very large number of competing substrates, e.g. those present in a combinatorial library, in a single reaction mixture has been developed in this contribution. The ratio of kcat/Km for isotopologs has also become a standard in mechanistic enzymology where kinetic isotope effects have been measured by the development of internal competition experiments with extreme precision. This contribution extends the theory of kinetic isotope effects to internal competition between three isotopologs present at non-tracer concentrations in the same reaction mix. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment.

Multiple mass isotopomer tracing of acetyl-CoA metabolism in Langendorff-perfused rat hearts: channeling of acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase.

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [(13)C2,(2)H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [(13)C6]glucose or [1,2-(13)C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-(13)C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.

Hidden specificity in an apparently nonspecific RNA-binding protein.

Nucleic-acid-binding proteins are generally viewed as either specific or nonspecific, depending on characteristics of their binding sites in DNA or RNA. Most studies have focused on specific proteins, which identify cognate sites by binding with highest affinities to regions with defined signatures in sequence, structure or both. Proteins that bind to sites devoid of defined sequence or structure signatures are considered nonspecific. Substrate binding by these proteins is poorly understood, and it is not known to what extent seemingly nonspecific proteins discriminate between different binding sites, aside from those sequestered by nucleic acid structures. Here we systematically examine substrate binding by the apparently nonspecific RNA-binding protein C5, and find clear discrimination between different binding site variants. C5 is the protein subunit of the transfer RNA processing ribonucleoprotein enzyme RNase P from Escherichia coli. The protein binds 5' leaders of precursor tRNAs at a site without sequence or structure signatures. We measure functional binding of C5 to all possible sequence variants in its substrate binding site, using a high-throughput sequencing kinetics approach (HITS-KIN) that simultaneously follows processing of thousands of RNA species. C5 binds different substrate variants with affinities varying by orders of magnitude. The distribution of functional affinities of C5 for all substrate variants resembles affinity distributions of highly specific nucleic acid binding proteins. Unlike these specific proteins, C5 does not bind its physiological RNA targets with the highest affinity, but with affinities near the median of the distribution, a region that is not associated with a sequence signature. We delineate defined rules governing substrate recognition by C5, which reveal specificity that is hidden in cellular substrates for RNase P. Our findings suggest that apparently nonspecific and specific RNA-binding modes may not differ fundamentally, but represent distinct parts of common affinity distributions.

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2'-O-transphosphorylation.

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2'-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the (18)O KIEs on the 2'O nucleophile, 5'O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium- or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5'O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/Lys41), together with proton transfer from His119, render departure of the 5'O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2'-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure.

Catalase abrogates ß-lapachone-induced PARP1 hyperactivation-directed programmed necrosis in NQO1-positive breast cancers.

Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. ß-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER-, PR-, Her2/Neu-)] breast cancers. To define cellular factors that influence the efficacy of ß-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where ∼120 moles of superoxide were formed per mole of ß-lapachone in 2 minutes. ß-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD(+)/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of ß-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. ß-Lapachone-induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of ß-lapachone and other NQO1 bioactivatable drugs.

Metabolomics and mass isotopomer analysis as a strategy for pathway discovery: pyrrolyl and cyclopentenyl derivatives of the pro-drug of abuse, levulinate.

We recently reported that levulinate (4-ketopentanoate) is converted in the liver to 4-hydroxypentanoate, a drug of abuse, and that the formation of 4-hydroxypentanoate is stimulated by ethanol oxidation. We also identified 3 parallel ß-oxidation pathways by which levulinate and 4-hydroxypentanoate are catabolized to propionyl-CoA and acetyl-CoA. We now report that levulinate forms three seven-carbon cyclical CoA esters by processes starting with the elongation of levulinyl-CoA by acetyl-CoA to 3,6-diketoheptanoyl-CoA. The latter γ-diketo CoA ester undergoes two parallel cyclization processes. One process yields a mixture of tautomers, i.e., cyclopentenyl- and cyclopentadienyl-acyl-CoAs. The second cyclization process yields a methyl-pyrrolyl-acetyl-CoA containing a nitrogen atom derived from the ε-nitrogen of lysine but without carbons from lysine. The cyclic CoA esters were identified in rat livers perfused with levulinate and in livers and brains from rats gavaged with calcium levulinate ± ethanol. Lastly, 3,6-diketoheptanoyl-CoA, like 2,5-diketohexane, pyrrolates free lysine and, presumably, lysine residues from proteins. This may represent a new pathway for protein pyrrolation. The cyclic CoA esters and related pyrrolation processes may play a role in the toxic effects of 4-hydroxypentanoate.

Metabolism of γ-hydroxybutyrate in perfused rat livers.

GHB (γ-hydroxybutyrate) is both a neurotransmitter and a drug of abuse (date-rape drug). We investigated the catabolism of this compound in perfused rat livers. Using a combination of metabolomics and mass isotopomer analysis, we showed that GHB is metabolized by multiple processes, in addition to its previously reported metabolism in the citric acid cycle via oxidation to succinate. A substrate cycle operates between GHB and γ-aminobutyrate via succinic semialdehyde. Also, GHB undergoes (i) ß-oxidation to glycolyl-CoA+acetyl-CoA, (ii) two parallel processes which remove C-1 or C-4 of GHB and form 3-hydroxypropionate from C-2+C-3+C-4 or from C-1+C-2+C-3 of GHB, and (iii) degradation to acetyl-CoA via 4-phosphobutyryl-CoA. The present study illustrates the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.

Peroxisome proliferator-activated receptor γ (PPARγ) mediates a Ski oncogene-induced shift from glycolysis to oxidative energy metabolism.

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through ß-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

Deconvolution of Raman spectroscopic signals for electrostatic, H-bonding, and inner-sphere interactions between ions and dimethyl phosphate in solution.

Quantitative analysis of metal ion-phosphodiester interactions is a significant experimental challenge due to the complexities introduced by inner-sphere, outer-sphere (H-bonding with coordinated water), and electrostatic interactions that are difficult to isolate in solution studies. Here, we provide evidence that inner-sphere, H-bonding and electrostatic interactions between ions and dimethyl phosphate can be deconvoluted through peak fitting in the region of the Raman spectrum for the symmetric stretch of non-bridging phosphate oxygen (ν(s)PO(2)(-)). An approximation of the change in vibrational spectra due to different interaction modes is achieved using ions capable of all or a subset of the three forms of metal ion interaction. Contribution of electrostatic interactions to ion-induced changes to the Raman ν(s)PO(2)(-) signal could be modeled by monitoring attenuation of ν(s)PO(2)(-) in the presence of tetramethylammonium, while contribution of H-bonding and inner-sphere coordination could be approximated from the intensities of altered ν(s)PO(2)(-) vibrational modes created by an interaction with ammonia, monovalent or divalent ions. A model is proposed in which discrete spectroscopic signals for inner-sphere, H-bonding, and electrostatic interactions are sufficient to account for the total observed change in ν(s)PO(2)(-) signal due to interaction with a specific ion capable of all three modes of interaction. Importantly, the quantitative results are consistent with relative levels of coordination predicted from absolute electronegativity and absolute hardness of alkali and alkaline earth metals.

Metabolism of levulinate in perfused rat livers and live rats: conversion to the drug of abuse 4-hydroxypentanoate.

Calcium levulinate (4-ketopentanoate) is used as an oral and parenteral source of calcium. We hypothesized that levulinate is converted in the liver to 4-hydroxypentanoate, a new drug of abuse, and that this conversion is accelerated by ethanol oxidation. We confirmed these hypotheses in live rats, perfused rat livers, and liver subcellular preparations. Levulinate is reduced to (R)-4-hydroxypentanoate by a cytosolic and a mitochondrial dehydrogenase, which are NADPH- and NADH-dependent, respectively. A mitochondrial dehydrogenase or racemase system also forms (S)-4-hydroxypentanoate. In livers perfused with [(13)C(5)]levulinate, there was substantial CoA trapping in levulinyl-CoA, 4-hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA. This CoA trapping was increased by ethanol, with a 6-fold increase in the concentration of 4-phosphopentanoyl-CoA. Levulinate is catabolized by 3 parallel pathways to propionyl-CoA, acetyl-CoA, and lactate. Most intermediates of the 3 pathways were identified by mass isotopomer analysis and metabolomics. The production of 4-hydroxypentanoate from levulinate and its stimulation by ethanol is a potential public health concern.

Cardiolipin: characterization of distinct oxidized molecular species.

Cardiolipin (CL) is a phospholipid predominantly found in the mitochondrial inner membrane and is associated structurally with individual complexes of the electron transport chain (ETC). Because the ETC is the major mitochondrial reactive oxygen species (ROS)-generating site, the proximity to the ETC and bisallylic methylenes of the PUFA chains of CL make it a likely target of ROS in the mitochondrial inner membrane. Oxidized cellular CL products, uniquely derived from ROS-induced autoxidation, could serve as biomarkers for the presence of the ROS and could help in the understanding of the mechanism of oxidative stress. Because major CL species have four unsaturated acyl chains, whereas other phospholipids usually have only one in the sn-2 position, characterization of oxidized CL is highly challenging. In the current study, we exposed CL, under aerobic conditions, to singlet oxygen (¹O2), the radical initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride, or room air, and the oxidized CL species were characterized by HPLC-tandem mass spectrometry (MS/MS). Our reverse-phase ion-pair HPLC-MS/MS method can characterize the major and minor oxidized CL species by detecting distinctive fragment ions associated with specific oxidized species. The HPLC-MS/MS results show that monohydroperoxides and bis monohydroperoxides were generated under all three conditions. However, significant amounts of CL dihydroperoxides were produced only by ¹O2-mediated oxidation. These products were barely detectable from radical oxidation either in a liposome bilayer or in thin film. These observations are only possible due to the chromatographic separation of the different oxidized species.

Binding to and photo-oxidation of cardiolipin by the phthalocyanine photosensitizer Pc 4.

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT.

Kinetic isotope effects for RNA cleavage by 2'-O- transphosphorylation: nucleophilic activation by specific base.

To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary (18)O kinetic isotope effects (KIEs) and solvent D(2)O isotope effects were measured to probe the mechanism of base-catalyzed 2'-O-transphosphorylation of the RNA dinucleotide 5'-UpG-3'. The observed (18)O KIEs for the nucleophilic 2'-O and in the 5'-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus-oxygen bond fission to the leaving group ((18)k(LG) = 1.034 +/- 0.004) and phosphorus-nucleophile bond formation ((18)k(NUC) = 0.984 +/- 0.004). A breakpoint in the pH dependence of the 2'-O-transphosphorylation rate to a pH independent phase above pH 13 has been attributed to the pK(a) of the 2'-OH nucleophile. A smaller nucleophile KIE is observed at pH 12 ((18)k(NUC) = 0.995 +/- 0.004) that is interpreted as the combined effect of the equilibrium isotope effect (ca. 1.02) on deprotonation of the 2'-hydroxyl nucleophile and the intrinsic KIE on the nucleophilic addition step (ca. 0.981). An alternative mechanism in which the hydroxide ion acts as a general base is considered unlikely given the lack of a solvent deuterium isotope effect above the breakpoint in the pH versus rate profile. These results represent the first direct analysis of the transition state for RNA strand cleavage. The primary (18)O KIE results and the lack of a kinetic solvent deuterium isotope effect together provide strong evidence for a late transition state and 2'-O nucleophile activation by specific base catalysis.

Photosensitization of intact heart mitochondria by the phthalocyanine Pc 4: Correlation of structural and functional deficits with cytochrome c release.

Singlet oxygen is produced by the absorption of red light by the phthalocyanine dye Pc 4, followed by energy transfer to dissolved triplet oxygen. Mitochondria preincubated with Pc 4 were illuminated by red light and the damage to mitochondrial structure and function by the generated singlet oxygen was studied. At early illumination times (3-5 min of red light exposure), State 3 respiration was inhibited (50%), whereas State 4 activity increased, resulting in effectively complete uncoupling. Individual complex activities were measured and only complex IV activity was significantly reduced and exhibited a dose response, whereas the activities of electron transport complexes I, II, and III were not significantly affected. Cytochrome c release was an increasing function of irradiation time, with 30% being released after 5 min of illumination. Mitochondrial expansion along with changes in the structure of the cristae were observed by transmission electron microscopy after 5 min of irradiation, with an increase in large vacuoles and membrane rupture occurring after more extensive exposures.

Photo-oxidation of cardiolipin and cytochrome c with bilayer-embedded Pc 4.

Singlet oxygen, (1)O(2), is produced by absorption of red light by the phthalocyanine dye Pc 4, followed by energy transfer to dissolved triplet molecular oxygen, (3)O(2). In tissues, Pc 4 concentrates in lipid bilayers, and particularly in mitochondrial membranes, because of its positive charge. Illumination of cells and tissues with red light after uptake of Pc 4 results in cell death. The potential initial chemical steps that result in cellular dysfunction have been characterized in this study. Both unsaturated acyl chains of phospholipids and proteins are identified as targets of oxidation. Tetra-linoleoyl cardiolipin was oxidized in both liposomes and mitochondria after Pc 4-mediated (1)O(2) generation. Evidence for the formation of both mono- and bis-hydroperoxide adducts of single linoleoyl side chains is provided by ESI-MS and ESI-MS/MS. Similarly, illumination of Pc 4 in liposomes and mitochondria resulted in cytochrome c oxidation as detected by oxidation of His 26 in the peptide H(26)*KTGPNLHGLFGK, further supporting the potential use of this peptide as a biomarker for the presence of mitochondrial oxidative stress characteristic of (1)O(2) in vivo (J. Kim et al., Free Radic. Biol. Med. 44:1700-1711; 2008). These observations provide evidence that formation of lipid hydroperoxides and/or protein oxidation can be the initial chemical steps in Pc 4-mediated induction of apoptosis in photodynamic therapy.