On the stoichiometry of the iron-sulphur clusters in mitochondrial NADH: ubiquinone oxidoreductase.

The concentration of the iron-sulphur (Fe-S) cluster 1b, present in complex I or soluble high-molecular-mass NADH dehydrogenase, was determined using different methods. It was found that direct double integration of the EPR signal at temperatures higher than 40 K, as is commonly used in this field of research, results in a considerable overestimation of the concentration of cluster 1b. It is demonstrated that this is caused by contributions from the relaxation-broadened signals of the Fe-S clusters 2-4 in the enzyme. The correct way for determining the intensity of the EPR signal of cluster 1b is by comparison with a simulated line shape. It is concluded that the concentration of cluster 1b is half that of cluster 2. This corroborates our proposal based on presteady-state kinetic and inhibitor-titration studies [Van Belzen, R., Van Gaalen, M. C. M., Cuypers, P. A. & Albracht S. P. J. (1990) Biochim. Biophys Acta 1017, 152-159] that the minimal functional unit of mitochondrial NADH:ubiquinone oxidoreductase must be a heterodimer.     

Spectroscopic characterization of the number and type of iron-sulfur clusters in NADH:ubiquinone oxidoreductase

The number and type of iron-sulfur clusters present in the NADH dehydrogenase of the mammalian respiratory chain were studied by a combination of low temperature magnetic circular dichroism (MCD) and quantitative electron paramagnetic resonance spectroscopies. MCD was used with the high molecular weight, soluble enzyme, and EPR was used with both the purified enzyme and Complex I (NADH:ubiquinone oxidoreductase). The results of the EPR experiments of the two types of preparations agreed with each other, as well as with the data in the literature for various types of membrane-bound preparations. The two methods gave concordant results showing the presence of one binuclear and of three tetranuclear NADH-reducible iron-sulfur clusters. Earlier studies using the cluster extrusion technique indicated a higher ratio of binuclear to tetranuclear clusters which may be explained by cluster interconversion during the extrusion process.     

Identification of the ubiquinone-binding site of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

In order to localize the ubiquinone-binding site of complex I (NADH:ubiquinone oxidoreductase), a novel photoreactive ubiquinone analogue (Q0C7ArN3) has been synthesized. It is shown that the direct chemical precursor of this analogue (Q0C7ArNO2) and the analogue itself are accepted as substrates in an enzyme assay utilizing ubiquinone-depleted mitochondrial membranes of Neurospora crassa. The activity of the enzyme applying these derivatives is inhibited by 50% at a concentration of 9 and 20 microM rotenone. Photoaffinity labeling experiments were performed with both isolated complex I and whole mitochondrial membranes of N. crassa under various conditions. In each of these experiments a protein subunit with an apparent molecular mass of about 9.5 kDa was labeled with high specificity. Radioactive labeling was totally prevented by the addition of ubiquinone-2 at concentrations higher than 500 microM but was not affected by comparable concentrations of rotenone or other hydrophobic substances. In the labeling experiments using whole membranes, the labeling signal was dramatically increased in the presence of 1.5 mM NADH. These results strongly suggest that the ubiquinone analogue interacts specifically with the enzyme.     

Reduction of the iron-sulfur clusters in mitochondrial NADH:ubiquinone oxidoreductase (complex I) by EuII-DTPA, a very low potential reductant

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the mitochondrial electron transport chain. It contains a flavin mononucleotide to oxidize NADH, and eight iron-sulfur clusters. Seven of them transfer electrons between the flavin and the quinone-binding site, and one is on the opposite side of the flavin. Although most information about their properties is from EPR, the spectra from only five clusters have been observed, and it is difficult to match them to the structurally defined clusters. Here, we analyze complex I from bovine mitochondria reacted with a very low potential reductant, to impose a potential approaching -1 V. We compare the spectra with those from higher potentials and from the 24 kDa subunit and flavoprotein subcomplex, and model the spectra by starting from those with fewer components and building the complexity gradually. Spectrum N1a, from the 24 kDa subunit [2Fe-2S] cluster, is not observed in bovine complex I at any potential. Spectrum N1b, from the 75 kDa subunit [2Fe-2S] cluster, exhibits a lower potential than the N3, N4 and N5 spectra of three [4Fe-4S] clusters. In the lowest potential spectra an N5-type spectrum is observed at unusually high temperature (indicating a significant change to the cluster, or that two clusters have very similar g values), the relaxation rate of N1b increases (indicating that a nearby cluster has become reduced) and a new feature with an apparent g value of 2.16 suggests an interaction between two reduced clusters. The consequences of these observations for electron transfer in complex I are discussed.     

Accumulation of the pre-assembled membrane arm of NADH:ubiquinone oxidoreductase in mitochondria of manganese-limited grown Neurospora crassa.

The NADH:ubiquinone oxidoreductase (complex I) of mitochondria is constructed from two arms arranged perpendicular to each other. The peripheral arm protruding into the matrix contains the proximal section of the electron pathway, and the membrane arm with all mitochondrially encoded subunits contains the distal section of the electron pathway. When Neurospora crassa is grown under manganese limitation the formation of the peripheral arm is disturbed, but the membrane arm containing the iron-sulfur cluster N-2, is accumulated. An extra-polypeptide, assumed to be a chaperone, is found to be associated with this pre-assembled membrane arm.     

Inhibition of mitochondrial NADH:ubiquinone oxidoreductase by ethoxyformic anhydride

The NADH:ubiquinone, but not the NADH:ferricyanide, reductase activity of mitochondrial complex I (NADH:ubiquinone oxidoreductase) is inhibited by incubation of the enzyme at pH 6.0 and 0 degree C with ethoxyformic anhydride (EFA), and the inhibition is partially reversed by subsequent incubation of EFA-treated complex I with hydroxylamine. These results and spectral changes of EFA-treated complex I in the u.v. region are consistent with modification of essential histidyl or tyrosyl residues between the primary NADH dehydrogenase and the site of ubiquinone reduction. Treatment of complex I with EFA in the presence of high concentrations of Seconal or Demerol did not protect against EFA inactivation, suggesting that the site of EFA modification may not be the same as the inhibiton sites of Seconal and Demerol. However, the presence of NADH during incubation of complex I with EFA greatly enhanced the inhibition rate, indicating that the reduced conformation of complex I is more susceptible to attack by EFA.     

A small isoform of NADH:ubiquinone oxidoreductase (complex I) without mitochondrially encoded subunits is made in chloramphenicol-treated Neurospora crassa

In mitochondria of Neurospora crassa grown in the presence of chloramphenicol a small form of NADH:ubiquinone reductase is made in place of the normal electron-transfer-complex I. This smaller enzyme has a molecular mass of approximately 350 kDa and consists of (at least) 13 different subunits which are all synthesized in the cytoplasm. The complex I which is normally found in Neurospora has a molecular mass of approximately 700 kDa and consists of around 30 different subunits, of which at least six are made in the mitochondria. Immunoblotting and peptide mapping suggest that the subunits of the small enzyme are homologous to subunits of the large enzyme, one subunit might even be identical. The small and the large NADH:ubiquinone reductases have the same high-affinity binding site for NADH but the two enzymes differ in the affinity and inhibitor sensitivity of the ubiquinone-binding site. The possibility is discussed that the small NADH:ubiquinone reductase is primitive isoform of complex I.     

Benzothiadiazole inhibits mitochondrial NADH:ubiquinone oxidoreductase in tobacco

An inducer of acquired disease resistance in plants, benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester, exhibited direct, concentration-dependent inhibition of the NADH:ubiquinone oxidoreductase activity of complex I of the mitochondrial electron transport chain of cultured tobacco cells. The complex I activity was less sensitive to inhibition by salicylic acid, an endogenous activator of acquired disease resistance. Using a dichlorodihydrofluorescein assay, it was found that benzothiadiazole, salicylic acid and the complex I inhibitor rotenone, increased reactive oxygen species production within cells in a concentration-dependent manner. The results indicate that both benzothiadiazole and salicylic acid affect the mitochondria of treated plant cells and result in increased production of reactive oxygen species. The biochemical basis of this response could be related to the inhibition of the NADH:ubiquinone oxidoreductase activity of complex I that results in channelling of electrons via complex II, with concomitant higher levels of superoxide production.     

De novo synthesis and desaturation of fatty acids at the mitochondrial acyl-carrier protein, a subunit of NADH:ubiquinone oxidoreductase in Neurospora crassa.

We have cultivated the cel mutant of Neurospora crassa defective in cytosolic fatty acid synthesis with [2-14C]malonate and found radioactivity covalently attached to the mitochondrial acyl-carrier protein (ACP), a subunit of the respiratory chain NADH:ubiquinone oxidoreductase. We purified the ACP by reverse-phase HPLC: the bound acyl groups were trans-esterified to methylesters and analyzed by gas chromatography. The saturated C6 to C18 fatty acids and oleic acid were detected. De novo synthesis and desaturation of fatty acids at the ACP subunit of NADH:ubiquinone oxidoreductase and use of the products of this mitochondrial synthetic pathway for cardiolipin synthesis is discussed.     

Characterization of the 9.5-kDa ubiquinone-binding protein of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

A small polypeptide subunit of the NADH:ubiquinone reductase (complex I) from Neurospora crassa has been identified by photoaffinity labeling to participate in the binding of ubiquinone [Heinrich, H., & Werner, S. (1992) Biochemistry (preceding paper in this issue)]. This polypeptide is further characterized by its primary structure and by an assessment of its localization within complex I. A lambda gt11 cDNA expression library was screened using a specific antibody directed against this individual subunit of complex I. Two groups of clones, coding for polypeptide subunits of the appropriate apparent molecular weight, were isolated. One group was shown to contain the relevant recombinants. The derived amino acid sequence for the 9.5-kDa ubiquinone-binding polypeptide shows a similarity with a putative ubiquinol-binding subunit (also a 9.5-kDa polypeptide) from complex III of bovine heart [Usui, S., Yu, L., & Tu, C.-A. (1990) Biochemistry 29, 4618-4626]. The polypeptide has a hydrophobic stretch of a sufficient length to span the membrane. It resists against extraction with NaBr or Na2CO3, and therefore probably is buried in the so-called hydrophobic membrane portion of complex I. This nuclearly-encoded subunit lacks a typical cleavable presequence and is imported into isolated mitochondria by a membrane potential-dependent process.     

Sensitivity to NN''-dicyclohexylcarbodi-imide of proton translocation by mitochondrial NADH:ubiquinone oxidoreductase.

Proton extrusion during ferricyanide reduction by NADH-generating substrates or succinate was studied in isolated rat liver mitochondria with the use of optical indicators. NN'-Dicyclohexylcarbodi-imide (DCCD) caused a decrease of 84% in the H+/e- ratio of NADH:cytochrome c reduction, but a decrease of only 49% in that of succinate:cytochrome c reduction, even though electron transfer was decreased equally in both spans. The data indicate that a DCCD-sensitive channel operates in the NADH:ubiquinone oxidoreductase region of the respiratory chain.     

EPR characterization of the iron-sulfur clusters in the NADH: ubiquinone oxidoreductase segment of the respiratory chain in Paracoccus denitrificans

The physicochemical properties of the iron-sulfur clusters present in the NADH:ubiquinone oxidoreductase of Paracoccus denitrificans have been examined in the cytoplasmic membrane particles by redox potentiometry and EPR spectroscopy. Analogous to the iron-sulfur clusters present in the mitochondrial NADH: ubiquinone oxidoreductase, we have found two binuclear and three tetranuclear EPR detectable iron-sulfur clusters, namely, N-1a, N-1b, N-2, N-3, and N-4. In the bacterial system, the two binuclear clusters differ in line shape and in Em values; the cluster with more rhombic symmetry (gx,y,z = 1.918, 1.937, 2.029) has the Em7.0 value of -150 while the almost axial one (gx,y,z = 1.929, 1.941, 2.019) has Em7.0 of -270 mV. The Em of the former cluster is pH dependent (-60 mV/pH) as in the case of mammalian N-1a while the latter is pH independent as is the mammalian cluster N-1b. The pH-dependent P. denitrificans [2Fe-2S] cluster, which we have labeled N-1a, has an Em7.0 as high as that of N-2, in contrast to the mammalian N-1a. Thus N-1a is reducible with a physiological reductant, NADH in this bacterial system. The Em of the cluster N-2 is also pH dependent (Em7.0 = -130 mV) with a pK value near 7.7. The Em values of all other clusters exhibit no pH dependence as in the case of their mammalian counterparts. We have found that the cluster N-1a is the most labile component among the five iron-sulfur clusters and may give rise to variable relative spin concentrations and extremely low Em values due to the facile modifications of the microenvironment of the cluster. The P. denitrificans NADH:ubiquinone oxidoreductase provides a unique and useful site I model system where redox composition is similar to the mitochondrial enzyme but with fewer numbers of polypeptides (Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311).     

Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa.

The rotenone sensitive NADH:ubiquinone was isolated from mitochondria of Neurospora crassa as a monodisperse preparation with the apparent mol. wt. in Triton solution of 0.9 X 10(6). The enzyme is composed of at least 22 subunits with apparent mol. wts. in SDS between 70 and 11 kd. Six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35S]methionine in the presence of cycloheximide. These subunits are synthesized in the mitochondria. Eleven subunits were radioactively labelled in the enzyme from cells which had incorporated [35S]methionine in the presence of chloramphenicol. These subunits are synthesized in the cytoplasm. The site of translation of the other subunits could not be established by the pulse-labelling technique. The assignment of the mitochondrially synthesized subunits to unidentified reading frames on the mitochondrial DNA is discussed.     

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

The first purification of bovine NADH:ubiquinone oxidoreductase (Complex I) was reported nearly half a century ago (Hatefi et al. J Biol Chem 237:1676–1680, 1962). The pathway of electron-transfer through the enzyme is still under debate. A major obstacle is the assignment of EPR signals to the individual iron-sulfur clusters in the subunits. The preceding paper described a working model based on the kinetics with NADPH. This model is at variance with current views in the field. The present paper provides a critical overview on the possible causes for the discrepancies. It is concluded that the stability of all purified preparations described thus far, including Hatefi’s Complex I, is compromised due to removal of the enzyme from the protective membrane environment. In addition, most preparations described during the last two decades are purified by methods involving synthetic detergents and column chromatography. This results in delipidation, loss of endogenous quinones and loss of reactions with (artificial) quinones in a rotenone-sensitive way. The Fe:FMN ratio’s indicate that FMN-a is absent, but that all Fe-S clusters may be present. In contrast to the situation in bovine SMP and Hatefi’s Complex I, three of the six expected [4Fe-4S] clusters are not detected in EPR spectra. Qualitatively, the overall EPR lineshape of the remaining three cubane signals may seem similar to that of Hatefi’s Complex I, but quantitatively it is not. It is further proposed that point mutations in any of the TYKY, PSST, 49-kDa or 30-kDa subunits, considered to make up the delicate structural heart of Complex I, may have unpredictable effects on any of the other subunits of this quartet. The fact that most point mutations led to inactive enzymes makes a correct interpretation of such mutations even more ambiguous. In none of the Complex-I-containing membrane preparations from non-bovine origin, the pH dependencies of the NAD(P)H→O₂ reactions and the pH-dependent reduction kinetics of the Fe-S clusters with NADPH have been determined. This excludes a proper discussion on the absence or presence of FMN-a in native Complex I from other organisms.     

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

Bovine NADH:ubiquinone oxidoreductase (Complex I) is the first complex in the mitochondrial respiratory chain. It has long been assumed that it contained only one FMN group. However, as demonstrated in 2003, the intact enzyme contains two FMN groups. The second FMN was proposed to be located in a conserved flavodoxin fold predicted to be present in the PSST subunit. The long-known reaction of Complex I with NADPH differs in many aspects from that with NADH. It was proposed that the second flavin group was specifically involved in the reaction with NADPH. The X-ray structure of the hydrophilic domain of Complex I from Thermus thermophilus (Sazanov and Hinchliffe 2006, Science 311, 1430–1436) disclosed the positions of all redox groups of that enzyme and of the subunits holding them. The PSST subunit indeed contains the predicted flavodoxin fold although it did not contain FMN. Inspired by this structure, the present paper describes a re-evaluation of the enigmatic reactions of the bovine enzyme with NADPH. Published data, as well as new freeze-quench kinetic data presented here, are incompatible with the general opinion that NADPH and NADH react at the same site. Instead, it is proposed that these pyridine nucleotides react at opposite ends of the 90?Å long chain of prosthetic groups in Complex I. Ubiquinone is proposed to react with the Fe-S clusters in the TYKY subunit deep inside the hydrophilic domain. A new model for electron transfer in Complex I is proposed. In the accompanying paper this model is compared with the one advocated in current literature.     

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane.

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (gz) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. The tetranuclear iron-sulfur clusters can have their gz axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.     

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain

We have studied the structure of bovine heart mitochondrial NADH:ubiquinone (Q) oxidoreductase (EC 1.6.99.3) by image analysis of electron micrographs. A three-dimensional reconstruction was calculated from a tilt-series of a two-dimensional crystal of the molecule. Our interpretation of the position of the molecule in the unit cell of the crystal is supported by additional (low-resolution) analysis of images of single molecules. The three-dimensional reconstruction was calculated with the aid of an iterative real-space reconstruction algorithm. The various projections used as input to the algorithm were obtained by averaging the images of the tilted crystal through a Fourier-space peak-filtering procedure. The reconstructed unit cell measures 15.2 X 15.2 nm in the plane of the two-dimensional crystal and has a height of 10-11 nm. The unit cell contains one molecule consisting of four large subunits. At the present resolution of about 1.3 nm in the untilted projection, these four monomers are seen as two dimers related by a two-fold axis. Two views of the single particles have been recognized; they are the top and side view of the building block of the crystal. After computer image alignment and correspondence analysis, clusters of similar particles have been averaged. In the averages an uneven stain distribution is seen around the molecules, which may result from preferential staining of hydrophilic parts of the molecule. The molecular mass of the whole molecule was determined from scanning transmission electron microscopy measurements as (1.6 +/- 0.2) X 10(6) daltons.     

The inhibition of mitochondrial complex I (NADH:ubiquinone oxidoreductase) by Zn2+

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, membrane-bound enzyme. It is central to energy transduction, an important source of cellular reactive oxygen species, and its dysfunction is implicated in neurodegenerative and muscular diseases and in aging. Here, we describe the effects of Zn2+ on complex I to define whether complex I may contribute to mediating the pathological effects of zinc in states such as ischemia and to determine how Zn2+ can be used to probe the mechanism of complex I. Zn2+ inhibits complex I more strongly than Mg2+, Ca2+, Ba2+, and Mn2+ to Cu2+ or Cd2+. It does not inhibit NADH oxidation or intramolecular electron transfer, so it probably inhibits either proton transfer to bound quinone or proton translocation. Thus, zinc represents a new class of complex I inhibitor clearly distinct from the many ubiquinone site inhibitors. No evidence for increased superoxide production by zinc-inhibited complex I was detected. Zinc binding to complex I is mechanistically complicated. During catalysis, zinc binds slowly and progressively, but it binds rapidly and tightly to the resting state(s) of the enzyme. Reactivation of the inhibited enzyme upon the addition of EDTA is slow, and inhibition is only partially reversible. The IC50 value for the Zn2+ inhibition of complex I is high (10-50 microm, depending on the enzyme state); therefore, complex I is unlikely to be a major site for zinc inhibition of the electron transport chain. However, the slow response of complex I to a change in Zn2+ concentration may enhance any physiological consequences.