Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. I. Pre-steady-state kinetics with NADPH

The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O₂ of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1–4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6–6.5.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. II. Kinetics of reoxidation of the reduced enzyme

The pre-steady-state kinetics of reoxidation of NADH:Q oxidoreductase present in submitochondrial particles has been studied by the freeze-quench method. It was found that at pH 8 only 50% of the Fe-S clusters 2 and 4 and 75% of the clusters 3 were rapidly reoxidised after transient and complete reduction by a pulse of NADH in the presence of excess NADPH. Thus, NADPH keeps 50% of the clusters 2 and 4 and 25% of the clusters 3 permanently reduced at this pH. Since NADH oxidation is nearly optimal at this pH, whereas NADPH oxidation is virtually absent, it was concluded that these permanently reduced clusters were not involved in the NADH oxidation activity. Incomplete reoxidation of the clusters 2, 3 and 4 after a pulse of NADH was also found in the absence of NADPH, both at pH 6.5 and at pH 8. A pulse of NADPH given at pH 6.5, where NADPH oxidation by oxygen is nearly optimal, caused a slow reduction of 50% of clusters 2 and 4 and 30% of the clusters 3, which persisted for a period of at least 15 s. It was concluded that these clusters were not involved in the oxidation of NADPH by oxygen, as catalysed by the particles. As a working hypothesis a dimeric model for NAD(P)H:Q oxidoreductase is proposed, consisting of two different protomers. One of the protomers, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, only reacts with NADH, and its oxidation by ubiquinone is rapid at pH but slow at pH 6.5. The other protomer, containing FMN and the clusters 2, 3 and 4, reacts with both NADH and NADPH and has a pH optimum at 6-6.5 for the reaction with ubiquinone.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. II. Kinetics of reoxidation of the reduced enzyme

The pre-steady-state kinetics of reoxidation of NADH:Q oxidoreductase present in submitochondrial particles has been studied by the freeze-quench method. It was found that at pH 8 only 50% of the Fe-S clusters 2 and 4 and 75% of the clusters 3 were rapidly reoxidised after transient and complete reduction by a pulse of NADH in the presence of excess NADPH. Thus, NADPH keeps 50% of the clusters 2 and 4 and 25% of the clusters 3 permanently reduced at this pH. Since NADH oxidation is nearly optimal at this pH, whereas NADPH oxidation is virtually absent, it was concluded that these permanently reduced clusters were not involved in the NADH oxidation activity. Incomplete reoxidation of the clusters 2, 3 and 4 after a pulse of NADH was also found in the absence of NADPH, both at pH 6.5 and at pH 8. A pulse of NADPH given at pH 6.5, where NADPH oxidation by oxygen is nearly optimal, caused a slow reduction of 50% of clusters 2 and 4 and 30% of the clusters 3, which persisted for a period of at least 15 s. It was concluded that these clusters were not involved in the oxidation of NADPH by oxygen, as catalysed by the particles. As a working hypothesis a dimeric model for NAD(P)H:Q oxidoreductase is proposed, consisting of two different protomers. One of the protomers, containing FMN and the Fe-S clusters 1–4 in stoichiometric amounts, only reacts with NADH, and its oxidation by ubiquinone is rapid at pH but slow at pH 6.5. The other protomer, containing FMN and the clusters 2, 3 and 4, reacts with both NADH and NADPH and has a pH optimum at 6–6.5 for the reaction with ubiquinone.     

The pathway of electron transfer in NADH:Q oxidoreductase

The pre-steady-state reduction by NADPH of NADH:Q oxidoreductase, as present in submitochondrial particles, has been further investigated with the rapid-mixing, rapid-freezing technique. It was found that trypsin treatment, that had previously been used to inactivate the transhydrogenase activity (Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Biophys. Acta 850, 413-422), considerably affected the stability at pH 6.2 of the NAD(P)H oxidation activity of submitochondrial particles. Use of the inhibitor butadione circumvented this problem, thus allowing a more careful investigation of the kinetics at pH 6.2. In the presence of the inhibitor rotenone it was found that 50% of the Fe-S clusters 3 and all of the Fe-S clusters 2 and 4 could be reduced by NADPH within 30 ms at pH 6.2. The remainder of the Fe-S clusters 3 and all of the Fe-S clusters 1 were reduced slowly (complete reduction only after more than 60 s). It was concluded that these latter Fe-S clusters play no role in the NADPH oxidation activity. In the absence of rotenone at pH 6.2 only 50% of the Fe-S clusters 2-4 could be reduced within 30 ms, while Fe-S cluster 1 was again not reduced. This difference was attributed to the fast reoxidation of part of the Fe-S clusters 2 and 4 by ubiquinone. At pH 8.0, where the NADPH oxidation activity is almost zero, 50% of the Fe-S clusters 2-4 could still be reduced by NADPH within 30 ms, while Fe-S cluster 1 was not reduced. The presence of rotenone had no effect on this reduction. From these observations it is concluded that the Fe-S clusters 2 and 4, which were rapidly reduced by NADPH and reoxidised by ubiquinone at pH 6.2, could not be reduced by NADPH at 8.0. This provides an explanation why NADH:Q oxidoreductase was not able to oxidise NADPH at pH 8.0, while part of the Fe-S clusters were still rapidly reduced. As a working hypothesis a dimeric structure for NADH:Q oxidoreductase is proposed. One protomer (B) contains FMN and Fe-S clusters 1-4 in equal amounts; the other protomer (A) is identical except for the absence of Fe-S cluster 1. NADH is able to react with both protomers, while NADPH only reacts with protomer A. A pH-dependent electron transfer from protomer A to protomer B is proposed, which would allow the reduction of Fe-S clusters 2 and 4 of protomer B by NADPH at pH 6.2, which is required for NADPH:Q oxidoreductase activity.     

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.     

Evidence that a type-2 NADH:quinone oxidoreductase mediates electron transfer to particulate methane monooxygenase in methylococcus capsulatus.

NADH readily provides reducing equivalents to membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) in isolated membrane fractions, but detergent solubilization disrupts this electron-transfer process. Addition of exogenous quinones (especially decyl-plastoquinone and duroquinone) restores the NADH-dependent pMMO activity. Results of inhibitor and substrate dependence of this activity indicate the presence of only a type-2 NADH:quinone oxidoreductase (NDH-2). A 100-fold purification of the NDH-2 was achieved using lauryl-maltoside solubilization followed by ion exchange, hydrophobic-interaction, and gel-filtration chromatography. The purified NDH-2 has a subunit molecular weight of 36 kDa and exists as a monomer in solution. UV-visible and fluorescence spectroscopy identified flavin adenine dinucleotide (FAD) as a cofactor present in stoichiometric amounts. NADH served as the source of electrons, whereas NADPH could not. The purified NDH-2 enzyme reduced coenzyme Q(0), duroquinone, and menaquinone at high rates, whereas the decyl analogs of ubiquinone and plastoquinone were reduced at approximately 100-fold lower rates. Rotenone and flavone did not inhibit the NDH-2, whereas amytal caused partial inhibition but only at high concentrations.     

Structure of NADH:Q oxidoreductase from bovine heart mitochondria studied by electron microscopy

Two-dimensional crystalline arrays of NADH:Q oxidoreductase preparations have been obtained by microdiffusion of protein dissolved in detergent against a 15 mM sodium acetate buffer of pH 5.5 containing 10% ($\text{w}\text{v}$) ammonium sulphate. Electron microscopy was used to study the structure of negatively stained crystals. Computer-reconstructed images were obtained by the Fourier peak filtering method. The crystals have p4 symmetry and a square unit cell with dimensions of 15.2 ± 0.5 nm. The four asymmetric units in the unit cell form a single tetrameric molecule with a dimension in the third direction of 8.2 nm. It is concluded on the basis of the estimated molecular mass that each tetramer cannot contain more than only one FMN molecule. This implies that the tetramers possibly are only a part of Complex I, since there is much evidence that one functional enzyme molecule of Complex I contains two FMN molecules.     

EPR signals of NADH: Q oxidoreductase. Shape and intensity.

(1) The EPR spectrum of Center 1 of NADH dehydrogenase in isolated Complex I or submitochondrial particles from beef heart consists of two overlapping nearly axial signals of the same intensity. They are defined as Center 1a (gll = 0.021, gl = 1.938) and Center 1b (gll = 2.021, gl = 1.928). (2) The line shape of the EPR spectrum of the Center 3+4 can be interpreted as an overlap of two rhombic signals of the same intensity. We define Center 3 by the g-values: gz=2.103, gy = 1.93-1.94, gx=1.884, and Center 4 by the values gz=2.04, gy=1.92-1.93, gx=1.863. (3) Direct quantitation of the individuals signals as well as computer stimulation suggests that the amount of the Centers 1a and 1b is only 25% of that of the other individuals centers and FMN. As EPR spectra of beef-heart submitochondrial particles at 10-20 K are nearly identical to those of Complex I, the same relative concentrations of the Fe-S centers are also present in the particles. (4) The signals either observed by us in EPR spectra of Complex I and submitochondrial particles at 4.2 K and high microwave powers can now be explained without assuming more than 5 paramagnetic centers in NADH dehydrogenase.     

Kinetic microplate-based assays for inhibitors of mitochondrial NADH:ubiquinone oxidoreductase (complex I) and succinate:cytochrome c oxidoreductase.

Kinetic microplate-based assays for both mitochondrial NADH:ubiquinone oxidoreductase (complex I) and succinate:cytochrome c oxidoreductase using insect submitochondrial particles as the source of the enzyme activities have been developed. These assays have been used to design high-throughput screens for inhibitors of these mitochondrial electron transfer activities to assess their intrinsic in vitro efficacies as potential pesticides. These methods can be used to test up to 60 compounds per day without the use of automated sample handling and diluting technology. The accuracy, specificity, and reproducibility of the microplate methods compared well with conventional spectrophotometer-based assays.     

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in bovine submitochondrial particles provide evidence for direct interaction between ATPase and NADH:Q oxidoreductase

From the chemiosmotic hypothesis it follows that no change is expected in potency of an uncoupler to inhibit an energy-driven reaction in an energy-transducing membrane if the energy-requiring part of the reaction, the so-called secondary proton pump, is partially inhibited by a specific, tightly bound inhibitor. An increase in potency upon inhibition of the primary pump may be expected, due to a lower rate of the total proton flow that can be used by the secondary pump and dissipated by the uncoupler. Contrary to this prediction several uncouplers (S13, SF6847, 2,4-dinitrophenol, valinomycin + nigericin) show an increase in uncoupling efficiency in ATP-driven reverse electron transfer (reversal) upon inhibition of the secondary pump in this reaction, the NADH:Q oxidoreductase, by rotenone. The increase in uncoupling efficiency is proportional to the decrease in the rate of reversal, that is to the decrease in concentration of active secondary pump. Similarly, upon inhibition of the primary pump, the ATPase, with oligomycin, an increase in uncoupling efficiency was found, also proportional to the decrease in the rate of reversal. When the pore-forming uncoupler gramicidin was used, no change in uncoupling potency was found upon inhibition of NADH:Q oxidoreductase. Inhibition of the ATPase, however, resulted in a proportionally lower uncoupling titre for gramicidin, just as was found for S13 in the presence of oligomycin. A difference was also found in the relative concentrations of S13 and gramicidin required to stimulate ATP hydrolysis or to inhibit reversal. The amount of S13 needed to stimulate ATP hydrolysis was clearly higher than the amount needed to inhibit reversal. On the contrary, the titre of gramicidin for both actions was about the same. To explain these results we propose that gramicidin uncouples via dissipation of the bulk delta mu H+, whereas the carrier-type uncouplers preferentially interfere with the direct energy transduction between the ATPase and redox enzymes. This is in accordance with the recently developed collision hypothesis.     

Mixed function oxidases in sterol metabolism. Separate routes for electron transfer from NADH and NADPH.

Oxidative deformylation of 4-hydroxy[14C]methylene-5alpha-cholest-7-en-3-one and oxidative demethylation of [30,31-14C]4,4-dimethyl-5alpha-cholest-7-en-3beta-ol by rat liver microsomes have been compared with regard to the manner in which electrons are introduced from both NADH and NADPH. Evidence suggests that NADH and NADPH support oxidation of both substrates via separate routes of electron transfer. Thus, 10 micron cytochrome c will inhibit NADPH-supported oxidation to 40 to 50% of control activity leaving NADH-supported oxidation unaffected. Also, treatment of microsomes with subtilisin diminishes NADPH-supported oxidation to 10 to 30% of control activity for either substrate to 70 to 90% of control activity while NADH-supported oxidative activity is virtually unaffected. Studies on the oxidase activities and NADPH-cytochrome c reductase as well as NADH-ferricyanide reductase have shown marked differences in activity in the presence of inhibitors. Thus, 9 mM 2'-AMP inhibits NADPH-cytochrome c reductase to 10 to 20% of control activity while NADPH-supported oxidative demethyl ation and deformylation are essentially unchanged. Mersalyl at 15 to 25 nmol/mg of microsomal protein inhibits both reductases to 20 to 40% of control activity; oxidative demethylation is unaffected and oxidative deformylation stimulated slightly when NADPH is used. Finally, antibody to NADPH-cytochrome c reductase inhibits oxidase activity for either substrate to 70 to 90% of control activity while reductase activity is inhibited to 10 to 30% of control activity.     

Evidence for two sites of superoxide production by mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).     

Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I)

The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site.     

A reductase/isomerase subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) carries an NADPH and is involved in the biogenesis of the complex.

Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I. The bacterial complex I consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. One of these extra subunits with a molecular mass of 40 kDa belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. We deleted this subunit in Neurospora crassa by gene disruption. In the mutant nuo 40, a complex I lacking the 40 kDa subunit is assembled. The mutant complex I does not contain tightly bound NADPH present in wild-type complex I. This NADPH cofactor is not connected to the respiratory electron pathway of complex I. The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-type complex I, one flavin mononucleotide and four iron-sulfur clusters detectable by electron paramagnetic resonance spectroscopy. In the mutant complex these groups are all readily reduced by NADH. However, the mutant complex is not capable of reducing ubiquinone. A recently described redox group identified in wild-type complex I by UV-visible spectroscopy is not detectable in the mutant complex. We propose that the reductase/isomerase subunit with its NADPH cofactor takes part in the biosynthesis of this new redox group.     

The iron-sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa.

Two related forms of the respiratory-chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear- and 6-7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only approximately 13 nuclear-encoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.     

Deactivation of mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I): Extrinsically affecting factors

Mitochondrial NADH:ubiquinone oxidoreductase (proton translocating respiratory complex I) serves several essential functions in cell metabolism: it maintains the intramitochondrial NADH/NAD⁺ ratio, contributes to generation of the proton-motive force, and participates in physiological and/or pathophysiological production of so-called reactive oxygen species. A characteristic feature of complex I is a slow, compared with its catalytic turnover, transformation to its inactive (deactivated) state, a phenomenon operationally called A/D transition. Here we report data on several extrinsic factors affecting deactivation as observed in coupled or uncoupled bovine heart submitochondrial particles. The time course of the strongly temperature-dependent deactivation deviates from first-order kinetics, and this deviation is abolished in the presence of an SH-group-specific reagent. The residual fraction of activity attained upon extensive deactivation shows the same kinetics of NADH oxidation as the fully active enzyme does. The rate of complex I deactivation is only slightly pH dependent within the range of 7.0–8.5 and significantly increases at higher pH. ATP∙(Mg) decreases the rate of complex I deactivation in coupled SMP, and this effect is abolished if the proton-motive force generating ATPase activity of F_o∙F₁ is precluded. Taken together, the data show that an equilibrium between the A and D forms of complex I exists. Possible mechanistic aspects of the deactivation process are discussed.     

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.     

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.     

Effect of the side chain structure of coenzyme Q on the steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase

Steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase using coenzyme Q with two isoprenoid unit (Q₂) or with a decyl group (DQ) show an ordered sequential mechanism in which the order of substrate binding and product release is NADH-Q₂ (DQ) -Q₂H₂ (DQH₂)-NAD⁺ in contrast to the order determined using Q₁ (Q₁-NADH-NAD⁺-Q₁H₂) (Nakashima et al., J. Bioenerg. Biomembr. 34, 11–19, 2002). The effect of the side chain structure of coenzyme Q suggests that NADH binding to the enzyme results in a conformational change, in the coenzyme Q binding site, which enables the site to accept coenzyme Q with a side chain significantly larger than one isoprenoid unit. The side chains of Q₂ and DQ bound to the enzyme induce a conformational change in the binding site to stabilize the substrate binding, while the side chain of Q₁ (one isoprenoid unit) is too short to induce the conformational change.