Stabilization of Enzymes by Using Thermophiles.

Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions. Metagenomic libraries from thermal environments are potential sources of thermostable enzymes of prokaryotic origin, but the number of expected hits could be quite low for enzymes handling substrates such as steroids, rarely found in prokaryotes. An alternative to metagenome screening is the selection of thermostable variants of well-known steroid-processing enzymes. Here we review and detail a protocol for such selection, where error-prone PCR (epPCR) is used to introduce random mutations into a gene to create a variants library for further selection of thermostable variants in the thermophile Thermus thermophilus. The method involves the use of folding interference vectors where the proper folding of the enzyme of interest at high temperature is linked to the folding of a reporter encoding a selectable property such as thermostable resistance to kanamycin, leading to a life-or-death selection of variants of reinforced folding independently of the activity of the enzyme.

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference.

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability" of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T m) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T m compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T m 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2.

Halohydrin dehalogenases (HHDHs) are promising enzymes for application in biocatalysis due to their promiscuous epoxide ring-opening activity with various anionic nucleophiles. So far, seven different HHDH subtypes A to G have been reported with subtype D containing the by far largest number of enzymes. Moreover, several characterized members of subtype D have been reported to display outstanding characteristics such as high catalytic activity, broad substrate spectra or remarkable thermal stability. Yet, no structure of a D-type HHDH has been reported to date that could be used to investigate and understand those features on a molecular level. We therefore solved the crystal structure of HheD2 from gamma proteobacterium HTCC2207 at 1.6 Å resolution and used it as a starting point for targeted mutagenesis in combination with molecular dynamics (MD) simulation, in order to study the low thermal stability of HheD2 in comparison with other members of subtype D. This revealed a hydrogen bond between conserved residues Q160 and D198 to be connected with a high catalytic activity of this enzyme. Moreover, a flexible surface region containing two α-helices was identified to impact thermal stability of HheD2. Exchange of this surface region by residues of HheD3 yielded a variant with 10 °C higher melting temperature and reaction temperature optimum. Overall, our results provide important insights into the structure-function relationship of HheD2 and presumably for other D-type HHDHs. DATABASES: Structural data are available in PDB database under the accession number 7B73.

Iodine-125 seeds in prostatic transurethral resection: Differential diagnosis with schistosome eggs / Presencia de I-125 semillas en material de resección transuretral. Diagnóstico diferencial con huevos de esquistosoma

The presence of foreign bodies in a prostate transurethral resection may pose a histopathological challenge. A 65-year-old white man with urinary obstructive symptoms was subjected to a transurethral resection. Histopathology showed a squamous cell carcinoma affecting prostatic ducts and multiple foreign bodies; their differential diagnosis was between iodine-125 seeds and Schistosoma sp. eggs, as both can show oval morphology and terminal spines. The seeds are irregular, homogeneous and solid, unlike Schistosoma eggs that are heterogeneous, with a lytic appearance and some embryonated or calcified. The seeds are located in prostatic ducts inducing periductal fibrosis whereas the Schistosoma sp. eggs are found in the stroma inducing desmoplasia and granulomas. The seeds are associated with a lymphoplasmacytic infiltrate while the eggs are surrounded by eosinophils

La presencia de cuerpos extraños en una resección transuretral de próstata puede suponer un desafío histopatológico. Un varón de 65 años con síntomas de obstrucción urinaria fue sometido a una resección transuretral. El estudio histopatológico demostró un carcinoma de células escamosas de conductos prostáticos y múltiples cuerpos extraños planteando el diagnóstico diferencial entre semillas de iodo-125 y huevos de esquistosoma. Ambos pueden tener morfología oval y espinas terminales. Las semillas son irregulares, homogéneas y sólidas, a diferencia de los huevos de esquistosoma que son heterogéneos, de apariencia lítica, y otros embrionados o calcificados. Las semillas se localizan en los ductos prostáticos induciendo fibrosis periductal mientras que los huevos están en el estroma induciendo desmoplasia y granulomas. Las semillas se asocian a infiltrado linfoplasmocítico mientras que los huevos están rodeados por eosinófilos

Iodine-125 seeds in prostatic transurethral resection: Differential diagnosis with schistosome eggs.

The presence of foreign bodies in a prostate transurethral resection may pose a histopathological challenge. A 65-year-old white man with urinary obstructive symptoms was subjected to a transurethral resection. Histopathology showed a squamous cell carcinoma affecting prostatic ducts and multiple foreign bodies; their differential diagnosis was between iodine-125 seeds and Schistosoma sp. eggs, as both can show oval morphology and terminal spines. The seeds are irregular, homogeneous and solid, unlike Schistosoma eggs that are heterogeneous, with a lytic appearance and some embryonated or calcified. The seeds are located in prostatic ducts inducing periductal fibrosis whereas the Schistosoma sp. eggs are found in the stroma inducing desmoplasia and granulomas. The seeds are associated with a lymphoplasmacytic infiltrate while the eggs are surrounded by eosinophils.

Diabody Pretargeting with Click Chemistry In Vivo.

UNLABELLED: Radioimmunotherapy and nuclear imaging (immuno-PET/SPECT) of cancer with radiometal-labeled antibody fragments or peptides is hampered by low tumor-to-kidney ratios because of high renal radiometal retention. Therefore, we developed and evaluated a pretargeting strategy using click chemistry in vivo to reduce kidney uptake and avoid unwanted radiation toxicity. We focused on the bioorthogonal reaction between a trans-cyclooctene (TCO)-functionalized TAG72 targeting diabody, AVP04-07, and a low-molecular-weight radiolabeled tetrazine probe that was previously shown to have low kidney retention and relatively fast renal clearance. METHODS: AVP04-07 diabodies were functionalized with TCO tags, and in vitro immunoreactivity toward bovine submaxillary mucin and tetrazine reactivity were assessed. Next, pretargeting biodistribution studies were performed in LS174T tumor-bearing mice with AVP04-07-TCO(n) (where n indicates the number of TCO groups per diabody) and radiolabeled tetrazine to optimize the TCO modification grade (0, 1.8, or 4.7 TCO groups per diabody) and the (177)Lu-tetrazine dose (0.1, 1.0, or 10 Eq with respect to the diabody). Radiolabeled tetrazine was injected at 47 h after diabody injection, and mice were euthanized 3 h later. A pretargeting SPECT/CT study with (111)In-tetrazine was performed with the optimized conditions. RESULTS: Immunoreactivity for native AVP04-07 was similar to that for TCO-functionalized AVP04-07, and the latter reacted efficiently with radiolabeled tetrazine in vitro. The combination of the pretargeting component AVP04-07 functionalized with 4.7 TCO groups and 1 Eq of (177)Lu-tetrazine with respect to the diabody showed the most promising biodistribution. Specifically, high (177)Lu-tetrazine tumor uptake (6.9 percentage injected dose/g) was observed with low renal retention, yielding a tumor-to-kidney ratio of 5.7. SPECT/CT imaging confirmed the predominant accumulation of radiolabeled tetrazine in the tumor and low nontumor retention. CONCLUSION: Pretargeting provides an alternative radioimmunotherapy and nuclear imaging strategy by overcoming the high renal retention of low-molecular-weight radiometal tumor-homing agents through the separate administration of a tumor-homing agent and a radioactive probe with fast clearance.

Trans-cyclooctene tag with improved properties for tumor pretargeting with the diels-alder reaction.

Radioimmunotherapy (RIT) of solid tumors is hampered by low tumor-to-nontumor (T/NT) ratios of the radiolabeled monoclonal antibodies resulting in low tumor doses in patients. Pretargeting technologies can improve the effectiveness of RIT in cancer therapy by increasing this ratio. We showed that a pretargeting strategy employing in vivo chemistry in combination with clearing agents, proceeds efficiently in tumor-bearing mice resulting in high T/NT ratios. A dosimetry study indicated that the chemical pretargeting technology, which centered on the bioorthogonal Diels-Alder click reaction between a radiolabeled tetrazine probe and a trans-cyclooctene-oxymethylbenzamide-tagged CC49 antibody (CC49-TCO(1)), can match the performance of clinically validated high-affinity biological pretargeting approaches in mice ( Rossin J Nucl Med. 2013 , 54 , 1989 - 1995 ). Nevertheless, the increased protein surface hydrophobicity of CC49-TCO(1) led to a relatively rapid blood clearance and concomitant reduced tumor uptake compared to native CC49 antibody. Here, we present the in vivo evaluation of a TCO-oxymethylacetamide-tagged CC49 antibody (CC49-TCO(2)), which is highly reactive toward tetrazines and less hydrophobic than CC49-TCO(1). CC49-TCO(2) was administered to healthy mice to determine its blood clearance and the in vivo stability of the TCO. Next, pretargeting biodistribution and SPECT studies with CC49-TCO(2), tetrazine-functionalized clearing agent, and radiolabeled tetrazine were carried out in nude mice bearing colon carcinoma xenografts (LS174T). CC49-TCO(2) had an increased circulation half-life, a 1.5-fold higher tumor uptake, and a 2.6-fold improved in vivo TCO stability compared to the more hydrophobic TCO-benzamide-CC49. As a consequence, and despite the 2-fold lower reactivity of CC49-TCO(2) toward tetrazines compared with CC49-TCO(1), administration of radiolabeled tetrazine afforded a significantly increased tumor accumulation and improved T/NT ratios in mice pretargeted with CC49-TCO(2). In conclusion, the TCO-acetamide derivative represents a large improvement in in vivo Diels-Alder pretargeting, possibly enabling application in larger animals and eventually humans.

Diels-Alder reaction for tumor pretargeting: in vivo chemistry can boost tumor radiation dose compared with directly labeled antibody.

UNLABELLED: Current pretargeting systems use noncovalent biologic interactions, which are prone to immunogenicity. We previously developed a novel approach based on the bioorthogonal reaction between a radiolabeled tetrazine and an antibody-conjugated trans-cyclooctene (TCO). However, the tumor-to-blood ratio was low due to reaction with freely circulating antibody-TCO. METHODS: Here we developed 2 tetrazine-functionalized clearing agents that enable rapid reaction with and removal of a TCO-tagged antibody (CC49) from blood. Next, we incorporated this approach into an optimized pretargeting protocol in LS174T-bearing mice. Then we compared the pretargeted (177)Lu-labeled tetrazine with (177)Lu-labeled CC49. The biodistribution data were used for mouse and human dosimetry calculations. RESULTS: The use of a clearing agent led to a doubling of the tetrazine tumor uptake and a 125-fold improvement of the tumor-to-blood ratio at 3 h after tetrazine injection. Mouse dosimetry suggested that this should allow for an 8-fold higher tumor dose than is possible with nonpretargeted radioimmunotherapy. Also, humans treated with CC49-TCO-pretargeted (177)Lu-tetrazine would receive a dose to nontarget tissues 1 to 2 orders of magnitude lower than with directly labeled CC49. CONCLUSION: The in vivo performance of chemical pretargeting falls within the range of results obtained for the clinically validated pretargeting approaches in mice, with the advantage of potentially allowing for fractionated radiotherapy as a result of a lower likelihood of immunogenicity. These findings demonstrate that biologic pretargeting concepts can be translated to rapid bioorthogonal chemical approaches with retained potential.

Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.

One of the challenges of pretargeted radioimmunotherapy, which centers on the capture of a radiolabeled probe by a preinjected tumor-bound antibody, is the potential immunogenicity of biological capturing systems. A bioorthogonal chemical approach may circumvent this drawback, but effective in vivo chemistry in mice, larger animals, and eventually humans, requires very high reagent reactivity, sufficient stability, and retained selectivity. We report here that the reactivity of the fastest bioorthogonal reaction, the inverse-electron-demand-Diels-Alder cycloaddition between a tetrazine probe and a trans-cyclooctene-tagged antibody, can be increased 10-fold (k2 = 2.7 × 10(5) M(-1) s(-1)) via the trans-cyclooctene, approaching the speed of biological interactions, while also increasing its stability. This was enabled by the finding that the trans-cyclooctene tag is probably deactivated through isomerization to the unreactive cis-cyclooctene isomer by interactions with copper-containing proteins, and that increasing the steric hindrance on the tag can impede this process. Next, we found that the higher reactivity of axial vs equatorial linked TCO can be augmented by the choice of linker. The new, stabilized, and more reactive tag allowed for improved tumor-to-nontumor ratios in pretargeted tumor-bearing mice.

SPECT/CT imaging of temperature-sensitive liposomes for MR-image guided drug delivery with high intensity focused ultrasound.

The goal of this study was to investigate the blood kinetics and biodistribution of temperature-sensitive liposomes (TSLs) for MR image-guided drug delivery. The co-encapsulated doxorubicin and [Gd(HPDO3A)(H2O)] as well as the ¹¹¹In-labeled liposomal carrier were quantified in blood and organs of tumor bearing rats. After TSL injection, mild hyperthermia (T=42 °C) was induced in the tumor using high intensity focused ultrasound under MR image-guidance (MR-HIFU). The biodistribution of the radiolabeled TSLs was investigated using SPECT/CT imaging, where the highest uptake of ¹¹¹In-labeled TSLs was observed in the spleen and liver. The MR-HIFU-treated tumors showed 4.4 times higher liposome uptake after 48 h in comparison with controls, while the doxorubicin concentration was increased by a factor of 7.9. These effects of HIFU-treatment are promising for applications in liposomal drug delivery to tumors.

L-arginine is a radioprotector for hematopoietic progenitor cells.

L-arginine is shown to protect hematopoietic progenitor (32D cl 3) cells from death due to exposure to γ radiation ((137)Cs). Some of the other intermediates in the urea cycle, namely ornithine and citrulline, plus urea itself, were not found to have any significant impact on cell survival after irradiation. Intriguingly, supplementation of irradiated cells with L-arginine results in decreased production of peroxynitrite, suggesting that suppression of superoxide generation by nitric oxide synthase in one or more microenvironments is an important factor in the observed radioprotection. The absence of any radioprotective effect of L-arginine in cells at 3% oxygen also confirms the involvement of one or more oxygen-derived species. Knockdown experiments with nitric oxide synthase (NOS) siRNAs in cells and NOS knockout animals confirm that the observed radioprotection is associated with nNOS (NOS-1). L-arginine also ameliorates the transient inhibition of the electron-transport chain complex I that occurs within 30 min of completing the dose (10 Gy) and that appears to be a functional marker for postirradiation mitochondrial oxidant production.

Block-copolymer-stabilized iodinated emulsions for use as CT contrast agents.

The objective of this study was to develop radiopaque iodinated emulsions for use as CT blood pool contrast agents. Three hydrophobic iodinated oils were synthesized based on the 2,3,5-triiodobenzoate moiety and formulated into emulsions using either phospholipids or amphiphilic polymers, i.e. Pluronic F68 and poly(butadiene)-b-poly(ethylene glycol) (PBD-PEO), as emulsifiers. The size, stability and cell viability was investigated for all stabilized emulsions. Three emulsions stabilized with either lipids or PBD-PEO were subsequently tested in vivo as a CT blood pool contrast agent in mice. While the lipid-stabilized emulsions turned out unstable in vivo, polymer-stabilized emulsions performed well in vivo. In blood, a contrast enhancement of 220 Hounsfield Units (HU) was measured directly after intravenous administration of 520 mg I/kg. The blood circulation half-life of a PBD-PEO stabilized emulsion was approximately 3 h and no noticeable in vivo toxicity was observed. These results show the potential of above emulsions for use as blood pool agents in contrast enhanced CT imaging.

Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance.

Local drug delivery of doxorubicin holds promise to improve the therapeutic efficacy and to reduce toxicity profiles. Here, we investigated the release of doxorubicin and [Gd(HPDO3A)(H(2)O)] from different temperature-sensitive liposomes for applications in temperature-induced drug delivery under magnetic resonance image guidance. In particular, two temperature-sensitive systems composed of DPPC:MPPC:DPPE-PEG2000 (low temperature-sensitive liposomes, LTSL) and DPPC:HSPC:cholesterol:DPPE-PEG2000 (traditional temperature-sensitive liposomes, TTSL) were investigated. The co-encapsulation of [Gd(HPDO3A)(H(2)O)], a clinically approved MRI contrast agent, did not influence the encapsulation and release of doxorubicin. The LTSL system showed a higher leakage of doxorubicin at 37 degrees C, but a faster release of doxorubicin at 42 degrees C compared to the TTSL system. Furthermore, the rapid release of both doxorubicin and the MRI contrast agent from the liposomes occurred near the melting phase transition temperature, making it possible to image the release of doxorubicin using MRI.

GATA4 and GATA5 are potential tumor suppressors and biomarkers in colorectal cancer.

PURPOSE: The transcription factors GATA4 and GATA5 are involved in gastrointestinal development and are inactivated by promoter hypermethylation in colorectal cancer. Here, we evaluated GATA4/5 promoter methylation as potential biomarkers for noninvasive colorectal cancer detection, and investigated the role of GATA4/5 in colorectal cancer. EXPERIMENTAL DESIGN: Promoter methylation of GATA4/5 was analyzed in colorectal tissue and fecal DNA from colorectal cancer patients and healthy controls using methylation-specific PCR. The potential function of GATA4/5 as tumor suppressors was studied by inducing GATA4/5 overexpression in human colorectal cancer cell lines. RESULTS: GATA4/5 methylation was observed in 70% (63/90) and 79% (61/77) of colorectal carcinomas, respectively, and was independent of clinicopathologic features. Methylation frequencies in normal colon tissues from noncancerous controls were 6% (5 of 88, GATA4; P < 0.001) and 13% (13 of 100, GATA5; P < 0.001). GATA4/5 overexpression suppressed colony formation (P < 0.005), proliferation (P < 0.001), migration (P < 0.05), invasion (P < 0.05), and anchorage-independent growth (P < 0.0001) of colorectal cancer cells. Examination of GATA4 methylation in fecal DNA from two independent series of colorectal cancer patients and controls yielded a sensitivity of 71% [95% confidence interval (95% CI), 55-88%] and specificity of 84% (95% CI, 74-95%) for colorectal cancer detection in the training set, and a sensitivity of 51% (95% CI, 37-65%) and specificity of 93% (95% CI, 84-100%) in the validation set. CONCLUSIONS: Methylation of GATA4/5 is a common and specific event in colorectal carcinomas, and GATA4/5 exhibit tumor suppressive effects in colorectal cancer cells in vitro. GATA4 methylation in fecal DNA may be of interest for colorectal cancer detection.

N-Myc downstream-regulated gene 4 (NDRG4): a candidate tumor suppressor gene and potential biomarker for colorectal cancer.

BACKGROUND: Identification of hypermethylated tumor suppressor genes in body fluids is an appealing strategy for the noninvasive detection of colorectal cancer. Here we examined the role of N-Myc downstream-regulated gene 4 (NDRG4) as a novel tumor suppressor and biomarker in colorectal cancer. METHODS: NDRG4 promoter methylation was analyzed in human colorectal cancer cell lines, colorectal tissue, and noncancerous colon mucosa by using methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. NDRG4 mRNA and protein expression were studied using real-time-PCR and immunohistochemistry, respectively. Tumor suppressor functions of NDRG4 were examined by colony formation, cell proliferation, and migration and invasion assays in colorectal cancer cell lines that were stably transfected with an NDRG4 expression construct. Quantitative methylation-specific PCR was used to examine the utility of NDRG4 promoter methylation as a biomarker in fecal DNA from 75 colorectal cancer patients and 75 control subjects. All P values are two-sided. RESULTS: The prevalence of NDRG4 promoter methylation in two independent series of colorectal cancers was 86% (71/83) and 70% (128/184) compared with 4% (2/48) in noncancerous colon mucosa (P < .001). NDRG4 mRNA and protein expression were decreased in colorectal cancer tissue compared with noncancerous colon mucosa. NDRG4 overexpression in colorectal cancer cell lines suppressed colony formation (P = .014), cell proliferation (P < .001), and invasion (P < .001). NDRG4 promoter methylation analysis in fecal DNA from a training set of colorectal cancer patients and control subjects yielded a sensitivity of 61% (95% confidence interval [CI] = 43% to 79%) and a specificity of 93% (95% CI = 90% to 97%). An independent test set of colorectal cancer patients and control subjects yielded a sensitivity of 53% (95% CI = 39% to 67%) and a specificity of 100% (95% CI = 86% to 100%). CONCLUSIONS: NDRG4 is a candidate tumor suppressor gene in colorectal cancer whose expression is frequently inactivated by promoter methylation. NDRG4 promoter methylation is a potential biomarker for the noninvasive detection of colorectal cancer in stool samples.

Promoter CpG island hypermethylation- and H3K9me3 and H3K27me3-mediated epigenetic silencing targets the deleted in colon cancer (DCC) gene in colorectal carcinogenesis without affecting neighboring genes on chromosomal region 18q21.

Chromosomal loss of 18q21 is a frequent event in colorectal cancer (CRC) development, suggesting that this region harbors tumor suppressor genes (TSGs). Several candidate TSGs, among which methyl-CpG-binding domain protein 1 (MBD1), CpG-binding protein CXXC1, Sma- and Mad-related protein 4 (SMAD4), deleted in colon cancer (DCC) and methyl-CpG-binding domain protein 2 (MBD2) are closely linked on a 4-Mb DNA region on chromosome18q21. As TSGs can be epigenetically silenced, this study investigates whether MBD1, CXXC1, SMAD4, DCC and MBD2 are subject to epigenetic silencing in CRC. Methylation-specific polymerase chain reaction and sodium bisulfite sequencing of these genes show that DCC, but not MBD1, CXXC1, SMAD4 and MBD2, has promoter CpG island methylation in CRC cell lines and tissues {normal mucosa [29.5% (18/61)], adenomas [81.0% (47/58)] and carcinomas [82.7% (62/75)] (P = 8.6 x 10(-9))} that is associated with reduced DCC expression, independent of 18q21 loss analyzed by multiplex ligation-dependent probe amplification. Reduced gene expression of CXXC1, SMAD4 and MBD2 correlates with 18q21 loss in CRC cell lines (P = 0.04, 0.02 and 0.02, respectively). Treatment with the demethylating agent 5-aza-2'-deoxycytidine, but not with the histone deacetylase inhibitor trichostatin A exclusively restored DCC expression in CRC cell lines. Chromatin immunoprecipitation studies reveal that the DCC promoter is marked with repressive histone-tail marks H3K9me3 and H3K27me3, whereas activity related H3K4me3 was absent. Only active epigenetic marks were detected for MBD1, CXXC1, SMAD4 and MBD2. This study demonstrates specific epigenetic silencing of DCC in CRC as a focal process not affecting neighboring genes on chromosomal region 18q21.

The resistance of electron-transport chain Fe-S clusters to oxidative damage during the reaction of peroxynitrite with mitochondrial complex II and rat-heart pericardium.

The effects of peroxynitrite and nitric oxide on the iron-sulfur clusters in complex II (succinate dehydrogenase) isolated from bovine heart have been studied primarily by EPR spectroscopy and no measurable damage to the constitutive 2Fe-2S, 3Fe-4S, or 4Fe-4S clusters was observed. The enzyme can be repeatedly oxidized with a slight excess of peroxynitrite and then quantitatively re-reduced with succinate. When added in large excess, peroxynitrite reacted with at least one tyrosine in each subunit of complex II to form 3-nitrotyrosines, but activity was barely compromised. Examination of rat-heart pericardium subjected to conditions leading to peroxynitrite production showed a small inhibition of complex II (16%) and a greater inhibition of aconitase (77%). In addition, experiments performed with excesses of sodium citrate and sodium succinate on rat-heart pericardium indicated that the "g approximately 2.01" EPR signal observed immediately following the beginning of conditions modeling oxidative/nitrosative stress, could be a consequence of both reversible oxidation of the constitutive 3Fe-4S cluster in complex II and degradation of the 4Fe-4S cluster in aconitase. However, the net signal envelope, which becomes apparent in less than 1min following the start of oxidative/nitrosative conditions, is dominated by the component arising from complex II. Taking into account the findings of a previous study concerning complexes I and III (L.L. Pearce, A.J. Kanai, M.W. Epperly, J. Peterson, Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors, Nitric Oxide 13 (2005) 254-263) it is now apparent that, with the exception of the cofactor in aconitase, mammalian (mitochondrial) iron-sulfur clusters are surprisingly resistant to degradation stemming from oxidative/nitrosative stress.