Biomedical subjects

Ulrich Brandt

Publications and source records attributed to Ulrich Brandt.

At least 37 records · Page 2Linked to original sources

PubMed2004

Histidine 129 in the 75-kDa subunit of mitochondrial complex I from Yarrowia lipolytica is not a ligand for [Fe4S4] cluster N5 but is required for catalytic activity.

Respiratory chain complex I contains 8-9 iron-sulfur clusters. In several cases, the assignment of these clusters to subunits and binding motifs is still ambiguous. To test the proposed ligation of the tetranuclear iron-sulfur cluster N5 of respiratory chain complex I, we replaced the conserved histidine 129 in the 75-kDa subunit from Yarrowia lipolytica with alanine. In the mutant strain, reduced amounts of fully assembled but destabilized complex I could be detected. Deamino-NADH: ubiquinone oxidoreductase activity was abolished completely by the mutation. However, EPR spectroscopic analysis of mutant complex I exhibited an unchanged cluster N5 signal, excluding histidine 129 as a cluster N5 ligand.

Amino Acid Sequence↗

PubMed2004

HDQ (1-hydroxy-2-dodecyl-4(1H)quinolone), a high affinity inhibitor for mitochondrial alternative NADH dehydrogenase: evidence for a ping-pong mechanism.

Alternative NADH dehydrogenases (NADH:ubiquinone oxidoreductases) are single subunit respiratory chain enzymes found in plant and fungal mitochondria and in many bacteria. It is unclear how these peripheral membrane proteins interact with their hydrophobic substrate ubiquinone. Known inhibitors of alternative NADH dehydrogenases bind with rather low affinities. We have identified 1-hydroxy-2-dodecyl-4(1H)quinolone as a high affinity inhibitor of alternative NADH dehydrogenase from Yarrowia lipolytica. Using this compound, we have analyzed the bisubstrate and inhibition kinetics for NADH and decylubiquinone. We found that the kinetics of alternative NADH dehydrogenase follow a ping-pong mechanism. This suggests that NADH and the ubiquinone headgroup interact with the same binding pocket in an alternating fashion.

Electron Transport Complex I↗

PubMed2004

5-Hydroxydecanoate is metabolised in mitochondria and creates a rate-limiting bottleneck for beta-oxidation of fatty acids.

5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K(+) (K(ATP)) channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA (5-HD-CoA), which is a substrate for the first step of beta-oxidation. We have now analysed the complete beta-oxidation of 5-HD-CoA using specially synthesised (and purified) substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared it with the metabolism of the physiological substrate decanoyl-CoA. At the second step of beta-oxidation, catalysed by enoyl-CoA hydratase, enzyme kinetics were similar using either decenoyl-CoA or 5-hydroxydecenoyl-CoA as substrate. The last two steps were investigated using l-3-hydroxyacyl-CoA dehydrogenase (HAD) coupled to 3-ketoacyl-CoA thiolase. V(max) for the metabolite of 5-HD (3,5-dihydroxydecanoyl-CoA) was fivefold slower than for the corresponding metabolite of decanoate (l-3-hydroxydecanoyl-CoA). The slower kinetics were not due to accumulation of d-3-hydroxyoctanoyl-CoA since this enantiomer did not inhibit HAD. Molecular modelling of HAD complexed with 3,5-dihydroxydecanoyl-CoA suggested that the 5-hydroxyl group could decrease HAD turnover rate by interacting with critical side chains. Consistent with the kinetic data, 5-hydroxydecanoyl-CoA alone acted as a weak substrate in isolated mitochondria, whereas addition of 100 mum 5-HD-CoA inhibited the metabolism of decanoyl-CoA or lauryl-carnitine. In conclusion, 5-HD is activated, transported into mitochondria and metabolised via beta-oxidation, albeit with rate-limiting kinetics at the penultimate step. This creates a bottleneck for beta-oxidation of fatty acids. The complex metabolic effects of 5-HD invalidate the use of 5-HD as a blocker of mitochondrial K(ATP) channels in studies of preconditioning.

Animals↗

PubMed2004

Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica.

Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.

Amino Acid Sequence↗

PubMed2004

Significance of respirasomes for the assembly/stability of human respiratory chain complex I.

We showed that the human respiratory chain is organized in supramolecular assemblies of respiratory chain complexes, the respirasomes. The mitochondrial complexes I (NADH dehydrogenase) and III (cytochrome c reductase) form a stable core respirasome to which complex IV (cytochrome c oxidase) can also bind. An analysis of the state of respirasomes in patients with an isolated deficiency of single complexes provided evidence that the formation of respirasomes is essential for the assembly/stability of complex I, the major entry point of respiratory chain substrates. Genetic alterations leading to a loss of complex III prevented respirasome formation and led to the secondary loss of complex I. Therefore, primary complex III assembly deficiencies presented as combined complex III/I defects. This dependence of complex I assembly/stability on respirasome formation has important implications for the diagnosis of mitochondrial respiratory chain disorders.

Cytochromes b↗

PubMed2004

Relaxation filtered hyperfine (REFINE) spectroscopy: a novel tool for studying overlapping biological electron paramagnetic resonance signals applied to mitochondrial complex I.

A simple strategy to separate overlapping electron paramagnetic resonance (EPR) signals in biological systems is presented. Pulsed EPR methods (inversion- and saturation-recovery) allow the determination of the T(1) spin-lattice relaxation times of paramagnetic centers. T(1) may vary by several orders of magnitude depending on the species under investigation. These variations can be employed to study selectively individual species from a spectrum that results from an overlap of two species using an inversion-recovery filtered (IRf) pulsed EPR technique. The feasibility of such an IRf field-swept technique is demonstrated on model compounds (alpha,gamma-bisphenylene-beta-phenylallyl-benzolate, BDPA, and 2,2,6,6-tetramethyl-piperidine-1-oxyl, TEMPO) and a simple strategy for the successful analysis of such mixtures is presented. Complex I is a multisubunit membrane protein of the respiratory chain containing several iron-sulfur (FeS) centers, which are observable with EPR spectroscopy. It is not possible to investigate the functionally important FeS cluster N2 separately because this EPR signal always overlaps with the other FeS signals. This cluster can be studied selectively using the IRf field-swept technique and its EPR spectrum is in excellent agreement with previous cw-EPR data from the literature. In addition, the possibility to separate the hyperfine spectra of two spectrally overlapping paramagnetic species is demonstrated by applying this relaxation filter together with hyperfine spectroscopy (REFINE). For the first time, the application of this filter to a three-pulse electron spin-echo envelope modulation (ESEEM) pulse sequence is demonstrated to selectively observe hyperfine spectra on a system containing two paramagnetic species. Finally, REFINE is used to assign the observed nitrogen modulation in complex I to an individual iron-sulfur cluster.

Allyl Compounds↗

PubMed2004

Synthesis and inhibitory action of novel acetogenin mimics with bovine heart mitochondrial complex I.

Studies on the inhibition mechanism of acetogenins, the most potent inhibitors of complex I, are useful to elucidate the structural and functional features of the terminal electron-transfer step of this enzyme. We synthesized acetogenin mimics that possess two alkyl tails without a gamma-lactone ring, named Deltalac-acetogenin, and examined their inhibitory action on bovine heart mitochondrial complex I. Unexpectedly, the Deltalac-acetogenin carrying two n-undecanyl groups (compound 3) elicited very potent inhibition comparable to that of bullatacin. The inhibitory potency of compound 3 markedly decreased with shortening the length of either or both alkyl tails, indicating that symmetric as well as hydrophobic properties of the inhibitor are important for the inhibition. Both acetylation and deoxygenation of either or both of two OH groups adjacent to the tetrahydrofuran (THF) rings resulted in a significant decrease in inhibitory potency. These structural dependencies of the inhibitory action of Deltalac-acetogenins are in marked contrast to those of ordinary acetogenins. Double-inhibitor titration of steady-state complex I activity showed that inhibition of compound 3 and bullatacin are not additive, though the inhibition site of both inhibitors is downstream of iron-sulfur cluster N2. Our results indicate that the mode of inhibitory action of Deltalac-acetogenins differs from that of ordinary acetogenins. Therefore, Deltalac-acetogenins can be regarded as a novel type of inhibitor acting on the terminal electron-transfer step of complex I.

Acetogenins↗

PubMed2004

Functional significance of conserved histidines and arginines in the 49-kDa subunit of mitochondrial complex I.

We have studied the ubiquinone-reducing catalytic core of NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica by a series of point mutations replacing conserved histidines and arginines in the 49-kDa subunit. Our results show that histidine 226 and arginine 141 probably do not ligate iron-sulfur cluster N2 but that exchanging these residues specifically influences the properties of this redox center. Histidines 91 and 95 were found to be essential for ubiquinone reductase activity of complex I. Mutations at the C-terminal arginine 466 affected ubiquinone affinity and inhibitor sensitivity but also destabilized complex I. These results provide further support for a high degree of structural conservation between the 49-kDa subunit of complex I and its ancestor, the large subunit of water-soluble [NiFe] hydrogenases. In several mutations of histidine 226, arginine 141, and arginine 466 the characteristic EPR signatures of iron-sulfur cluster N2 became undetectable, but specific, inhibitor-sensitive ubiquinone reductase activity was only moderately reduced. As we could not find spectroscopic indications for a modified cluster N2, we concluded that these complex I mutants were lacking most of this redox center but were still capable of catalyzing inhibitor-resistant ubiquinone reduction at near normal rates. We discuss that this at first surprising scenario may be explained by electron transfer theory; after removal of a single redox center in a chain, electron transfer rates are predicted to be still much faster than steady-state turnover of complex I. Our results question some of the central mechanistic functions that have been put forward for iron-sulfur cluster N2.

Amino Acid Sequence↗

PubMed2004

Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species.

Dihydrocalcein (H2-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H2-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H2-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5 min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H2-calcein but increased the oxidation of H2-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H2-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H2-DCF. In contrast, DPI inhibited H2-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H2-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H2-calcein, but not H2-DCF inhibited complex I activity. These observations indicate that H2-DCF is an indicator for intracellular ROS, whereas the oxidation of H2-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.

Angiotensin II↗

PubMed2004

Processing of the 24 kDa subunit mitochondrial import signal is not required for assembly of functional complex I in Yarrowia lipolytica.

A small deletion in the second intron of human NDUFV2 (IVS2+5_+8delGTAA) has been shown to cause hypertrophic cardiomyopathy and encephalomyopathy [Bénit, P., Beugnot, R., Chretien, D., Giurgea, I., de Lonlay-Debeney, P., Issartel, J.P., Kerscher, S., Rustin, P., Rötig, A. & Munnich, A. (2003) Human Mutat.21, 582-586]. Skipping of exon 2 results in a partial deletion of the mitochondrial targeting sequence of the precursor for the 24 kDa subunit of respiratory chain complex I. Immunoreactivity of the 24 kDa subunit and complex I activity, both present at 30-50% of normal levels in patient mitochondria, raised the question of how the mutant 24 kDa subunit precursor can be imported and assembled into functional complex I. In the present study, we have remodelled the human NDUFV2 mutation by deleting codons 17-32 from the orthologous NUHM gene of the obligate aerobic yeast Yarrowia lipolytica. The resulting mutant enzyme was indistinguishable from parental complex I with regard to activity, inhibitor sensitivity and EPR signature. Size, isoelectric point and presumably also N-terminal acetylation were altered, indicating that the residual targeting sequence was retained on the mature 24 kDa protein. Complete removal of the NUHM presequence resulted in the absence of complex I activity, strongly arguing against the presence of an internal mitochondrial targeting sequence within the 24 kDa protein.

Amino Acid Sequence↗

PubMed2003

Cardiolipin stabilizes respiratory chain supercomplexes.

Cardiolipin stabilized supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase, respectively), but was not essential for their formation in the inner mitochondrial membrane because they were found also in a cardiolipin-deficient strain. Reconstitution with cardiolipin largely restored wild-type stability. The putative interface of complexes III and IV comprises transmembrane helices of cytochromes b and c1 and tightly bound cardiolipin. Subunits Rip1p, Qcr6p, Qcr9p, Qcr10p, Cox8p, Cox12p, and Cox13p and cytochrome c were not essential for the assembly of supercomplexes; and in the absence of Qcr6p, the formation of supercomplexes was even promoted. An additional marked effect of cardiolipin concerns cytochrome c oxidase. We show that a cardiolipin-deficient strain harbored almost inactive resting cytochrome c oxidase in the membrane. Transition to the fully active pulsed state occurred on a minute time scale.

ATP Synthetase Complexes↗

PubMed2003

Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity.

Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.

Amino Acid Substitution↗

PubMed2003

Mitochondrial complex I is deficient in renal oncocytomas.

Renal oncocytomas are benign tumors characterized by dense accumulation of mitochondria the cause of which remains unknown so far. Consistently, mitochondrial DNA content and the amounts and catalytic activities of several oxidative phosphorylation (OXPHOS) complexes were known to be increased in these tumors, but it was not ascertained that the OXPHOS system was functional. Here we investigated mitochondrial complex I and found that its NADH dehydrogenase activity and protein content were specifically decreased in oncocytomas, in stark contrast with the parallel decrease of all respiratory chain complexes in other, malignant, renal tumors. We conclude that deficiency of complex I in oncocytomas might be the early event causing the increased mitochondrial biogenesis, attempting to compensate for the loss of OXPHOS function. Since other tumors were found to be linked to mitochondrial deficiencies like genetic alterations of fumarate hydratase or succinate dehydrogenase, oncocytoma could be the third type of benign tumor associated with impairment of mitochondrial ATP production in an oxidative, quiescent tissue. Besides, complex I enzyme activity was moderately decreased in the vicinity of oncocytomas, when compared with normal tissue adjacent to other renal tumors. This suggested that oncocytomas are the result of at least two serial modifications altering the mitochondrial respiratory chain.

Adenoma, Oxyphilic↗

PubMed2003

Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron-sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this 'catalytic core' region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.

Amino Acid Sequence↗

PubMed2003

Functional implications from an unexpected position of the 49-kDa subunit of NADH:ubiquinone oxidoreductase.

Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer.

Amino Acid Sequence↗

PubMed2002

Halothane, isoflurane and sevoflurane inhibit NADH:ubiquinone oxidoreductase (complex I) of cardiac mitochondria.

We have investigated the effects of volatile anaesthetics on electron transport chain activity in the mammalian heart. Halothane, isoflurane and sevoflurane reversibly increased NADH fluorescence (autofluorescence) in intact ventricular myocytes of guinea-pig, suggesting that NADH oxidation was impaired. Using pig heart submitochondrial particles we found that the anaesthetics dose-dependently inhibited NADH oxidation in the order: halothane > isoflurane = sevoflurane. Succinate oxidation was unaffected by either isoflurane or sevoflurane, indicating that these agents selectively inhibit complex I (NADH:ubiquinone oxidoreductase). In addition to inhibiting NADH oxidation, halothane also inhibited succinate oxidation (and succinate dehydrogenase), albeit to a lesser extent. To test the hypothesis that complex I is a target of volatile anaesthetics, we examined the effects of these agents on NADH:ubiquinone oxidoreductase (EC 1.6.99.3) activity using the ubiquinone analogue DBQ (decylubiquinone) as substrate. Halothane, isoflurane and sevoflurane dose-dependently inhibited NADH:DBQ oxidoreductase activity. Unlike the classical inhibitor rotenone, none of the anaesthetics completely inhibited enzyme activity at high concentration, suggesting that these agents bind weakly to the 'hydrophobic inhibitory site' of complex I. In conclusion, halothane, isoflurane and sevoflurane inhibit complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain. At concentrations of approximately 2 MAC (minimal alveolar concentration), the activity of NADH:ubiquinone oxidoreductase was reduced by about 20 % in the presence of halothane or isoflurane, and by about 10 % in the presence of sevoflurane. These inhibitory effects are unlikely to compromise cardiac performance at usual clinical concentrations, but may contribute to the mechanism by which volatile anaesthetics induce pharmacological preconditioning.

Anesthetics, Inhalation↗

PubMed2002

Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids.

NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 micromol NADH min(-1) mg(-1) were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.

Electron Transport Complex I↗

PubMed2002

Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I.

The obligate aerobic yeast Yarrowia lipolytica is introduced as a powerful new model for the structural and functional analysis of mitochondrial complex I. A brief introduction into the biology and the genetics of this nonconventional yeast is given and the relevant genetic tools that have been developed in recent years are summarized. The respiratory chain of Y. lipolytica contains complexes I-IV, one "alternative" NADH-dehydrogenase (NDH2) and a non-heme alternative oxidase (AOX). Because the NADH binding site of NDH2 faces the mitochondrial intermembrane space rather than the matrix, complex I is an essential enzyme in Y. lipolytica. Nevertheless, complex I deletion strains could be generated by attaching the targeting sequence of a matrix protein, thereby redirecting NDH2 to the matrix side. Deletion strains for several complex I subunits have been constructed that can be complemented by shuttle plasmids carrying the deleted gene. Attachment of a hexa-histidine tag to the NUGM (30 kDa) subunit allows fast and efficient purification of complex I from Y. lipolytica by affinity-chromatography. The purified complex has lost most of its NADH:ubiquinone oxidoreductase activity, but is almost fully reactivated by adding 400-500 molecules of phosphatidylcholine per complex I. The established set of genetic tools has proven useful for the site-directed mutagenesis of individual subunits of Y. lipolytica complex I. Characterization of a number of mutations already allowed for the identification of several functionally important amino acids, demonstrating the usefulness of this approach.

Electron Spin Resonance Spectroscopy↗