Large-scale column-free purification of bovine F-ATP synthase.

Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers, and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability, and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring inhibitor factor 1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin to lauryl maltose neopentyl glycol ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.

Modified Blue Native Gel Approach for Analysis of Respiratory Supercomplexes.

In mitochondrial oxidative phosphorylation (Ox-Phos), individual electron transport chain complexes are thought to assemble into supramolecular entities termed supercomplexes (SCs). The technique of blue native (BN) gel electrophoresis has emerged as the method of choice for analyzing SCs. However, the process of sample extraction for BN gel analysis is somewhat tedious and introduces the possibility for experimental artifacts. Here we outline a streamlined method that eliminates a centrifugation step and provides a more representative sampling of a population of mitochondria on the final gel. Using this method, we show that SC composition does not appear to change dynamically with altered mitochondrial function.

A Method for Analysis of Nitrotyrosine-Containing Proteins by Immunoblotting Coupled with Mass Spectrometry.

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.

Bridging the Gap Between Nature and Antioxidant Setbacks: Delivering Gallic Acid to Mitochondria.

Research on mitochondria-targeted active molecules became a hot topic in the past decade. Development of mitochondria permeability transition pore (mPTP )-targeting agents with clinical applications is needed not only because of the importance of the target in several diseases but also due to the fact that the current developed molecules have shown poor clinical success. In fact, only a reduced percentage reach mitochondria , effectively preventing pathological mPTP opening. The mitochondrial-targeting strategies should be a promising solution to increase the selectivity of compounds to the mPTP , reducing also their potential side effects. Chemical conjugation of bioactive molecules with a lipophilic cation such as the triphenylphosphonium (TPP +) has been established as a robust strategy to specifically target mitochondria . Phytochemicals such as hydroxybenzoic acids are normal constituents of the human diet. These molecules display beneficial healthy effects, ranging from antioxidant action through diverse mechanisms to modulation of mitochondrial-related apoptotic system, although their therapeutic application is limited due to pharmacokinetic drawbacks. Accordingly, the development of a new antioxidant based on the dietary benzoic acid-gallic acid -is described as well as the demonstration of its mitochondriotropic characteristics.

Ca2+ as cofactor of the mitochondrial H+ -translocating F1 FO -ATP(hydrol)ase.

The mitochondrial F1 FO -ATPase in the presence of the natural cofactor Mg2+ acts as the enzyme of life by synthesizing ATP, but it can also hydrolyze ATP to pump H+ . Interestingly, Mg2+ can be replaced by Ca2+ , but only to sustain ATP hydrolysis and not ATP synthesis. When Ca2+ inserts in F1 , the torque generation built by the chemomechanical coupling between F1 and the rotating central stalk was reported as unable to drive the transmembrane H+ flux within FO . However, the failed H+ translocation is not consistent with the oligomycin-sensitivity of the Ca2+ -dependent F1 FO -ATP(hydrol)ase. New enzyme roles in mitochondrial energy transduction are suggested by recent advances. Accordingly, the structural F1 FO -ATPase distortion driven by ATP hydrolysis sustained by Ca2+ is consistent with the permeability transition pore signal propagation pathway. The Ca2+ -activated F1 FO -ATPase, by forming the pore, may contribute to dissipate the transmembrane H+ gradient created by the same enzyme complex.

Identification of unusual phospholipids from bovine heart mitochondria by HPLC-MS/MS.

Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated MS/MS spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species, such as PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids, including minor species.

Free-Radical-Mediated Formation of Trans-Cardiolipin Isomers, Analytical Approaches for Lipidomics and Consequences of the Structural Organization of Membranes.

Free-radical-mediated processes, such as peroxidation, isomerization and hydrogenation affecting fatty acid integrity and biological functions, have a trans-disciplinary relevance. Cardiolipins (CL, (1,3-diphosphatidyl-sn-glycerol)) and tetra-linoleoyl-CL are complex phospholipids, exclusively present in the Inner Mitochondrial Membrane (IMM) lipids, where they maintain membrane integrity and regulate enzyme functionalities. Peroxidation pathways and fatty acid remodeling are known causes of mitochondrial disfunctions and pathologies, including cancer. Free-radical-mediated isomerization with the change of the cis CL into geometrical trans isomers is an unknown process with possible consequences on the supramolecular membrane lipid organization. Here, the formation of mono-trans CL (MT-CL) and other trans CL isomers (T-CL) is reported using CL from bovine heart mitochondria and thiyl radicals generated by UV-photolysis from 2-mercaptoethanol. Analytical approaches for CL isomer separation and identification via 1H/13C NMR are provided, together with the chemical study of CL derivatization to fatty acid methyl esters (FAME), useful for lipidomics and metabolomics research. Kinetics information of the radical chain isomerization process was obtained using γ-irradiation conditions. The CL isomerization affected the structural organization of membranes, as tested by the reduction in unilamellar liposome diameter, and accompanied the well-known process of oxidative consumption induced by Fenton reagents. These results highlight a potential new molecular modification pathway of mitochondrial lipids with wide applications to membrane functions and biological consequences.

Toxicity of lupane derivatives on anionic membrane models, isolated rat mitochondria and selected human cell lines: Role of terminal alkyl chains.

Triterpenoids have multiple biological properties, although little information is available regarding their toxicity. The present study evaluates the toxicity of two new synthetic lupane derivatives using distinct biological models including synthetic lipids membranes, isolated liver and heart mitochondria fractions, and cell lines in culture. The two novel triterpenoids caused perturbations in the organization of synthetic lipid bilayers, leading to changes in membrane fluidity. Inhibition of cell proliferation and mitochondrial and nuclear morphological alterations were also identified. Inhibition of mitochondrial oxygen consumption, transmembrane electric potential depolarization and induction of the mitochondrial permeability transition pore was observed, although effects on isolated mitochondrial fractions were tissue-dependent (e.g. liver vs. heart). The size and length of hydrocarbon chains in the two molecules appear to be determinant for the degree of interaction with mitochondria, especially in the whole cell environment, where more barriers for diffusion exist. The results suggest that the positively charged triterpenoids target mitochondria and disrupt bioenergetics.

Mitochondrial complex I in the post-ischemic heart: reperfusion-mediated oxidative injury and protein cysteine sulfonation.

A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of O2-. The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to injury. Herein we employed LC-MS/MS to identify protein sulfonation of complex I in mitochondria from the infarct region of rat hearts subjected to 30-min of coronary ligation and 24-h of reperfusion in vivo as well as the mitochondria of sham controls. Mitochondrial preparations from the I/R regions had enhanced sulfonation levels on the cysteine ligands of iron­sulfur clusters, including N3 (C425), N1b (C92), N4 (C226), N2 (C158/C188), and N1a (C134/C139). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus sulfonation of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and an antioxidative function. Increased C134/C139 sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O2- generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C125, C187, C206, C238, C255, C286), 75 kDa (C367, C554, C564, C727), 49 kDa (C146, C326, C347), and PSST (C188). These results were consistent with the consensus indicating that 51 kDa and 75 kDa are two of major subunits hosting regulatory thiols, and their enhanced sulfonation by I/R predisposed the myocardium to further oxidant stress with impaired ubiquinone reduction. MS analysis further showed I/R-mediated enhanced sulfonation at the supernumerary subunits of 42 kDa (C67, C112, C183, C253), 15 kDa (C43), and 13 kDa (C79). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C43 of the 15 kDa subunit forms an intramolecular disulfide bond with C56, which was reported to stabilize complex I structure. C79 of the 13 kDa subunit is involved in Zn2+-binding, which was reported functionally important for complex I assembly. C79 sulfonation by I/R was found to impair Zn2+-binding. No significant enhancement of protein sulfonation was observed in mitochondrial complex I from the rat heart subjected to 30-min ischemia alone in vivo despite a decreased state 3 respiration, suggesting that the physiologic conditions of hyperoxygenation during reperfusion mediated an increase in complex I sulfonation and oxidative injury. In conclusion, sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increasing •O2- production, and mediating redox dysfunction of complex I.

Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy-transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ∼20 Šabove the membrane surface. Structural data suggest it accesses the site by a narrow channel, long enough to accommodate almost all of its ∼50-Šisoprenoid chain. However, how ubiquinone/ubiquinol exchange occurs on catalytically relevant timescales, and whether binding/dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly charged region at isoprenoids 4-7. We demonstrate that ubiquinol-10 dissociation is not rate determining and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion.

Influences of ethanol on the structure of toxic trans-crotonaldehyde in mitochondria coming from rat myocardium.

Inappropriate use of ethanol (EtOH) had led to noticeable health problems, but a beneficial phenomenon was found that EtOH displayed unique influences for toxic trans-crotonaldehyde (TCA) derived from mitochondrial lipid peroxidation. The influences of EtOH on the structure of TCA were systematically probed by UV-vis & Raman spectroscopy in the absence and presence of mitochondria, respectively. The maximum UV-vis peak at 301 nm of TCA was red shifted by hydroxyl (-OH) and methyl (-CH3) of EtOH, respectively. Raman stretching band of aldehyde (-CH=O) of TCA (TCA-CH=O) was split by the -CH3 of EtOH. The -CH3 increased TCA-CH=O stretching frequency while the -OH induced it. The more exposed -OH, the less stretching frequency. The ectopic -CH3 red shifted the UV-vis peak at 301 nm and Raman band of TCA-CH=O. In mitochondria, EtOH red shifted Raman stretching band of TCA-CH=O. Raman stretching bands of C-H, C-O and C-C of EtOH were red shifted, while Raman stretching bands of -CH2 and C-C-O of EtOH disappeared. The paper unearths the influences of EtOH to trap and transform the structure of TCA-CH=O. This discovery has an important contribution to eliminate TCA in order to protect and repair mtDNA by means of the decrease of 8-oxoG.

Mitochondrial transition ROS spike (mTRS) results from coordinated activities of complex I and nicotinamide nucleotide transhydrogenase.

Mitochondria exhibit suppressed ATP production, membrane potential (∆Ψmt) polarization and reactive oxygen species (ROS) bursts during some cellular metabolic transitions. Although mitochondrial ROS release is influenced by ∆Ψmt and respiratory state, the relationship between these properties remains controversial primarily because they have not been measured simultaneously. We developed a multiparametric method for probing mitochondrial function that allowed precise characterization of the temporal relationship between ROS, ∆Ψmt and respiration. We uncovered a previously unknown spontaneous ROS spike - termed mitochondrial transition ROS spike (mTRS) - associated with re-polarization of ∆Ψmt that occurs at the transition between mitochondrial energy states. Pharmacological inhibition of complex CI (CI), nicotinamide nucleotide transhydrogenase (NNT) and antioxidant system significantly decreased the ability of mitochondria to exhibit mTRS. NADH levels followed a similar trend to that of ROS during the mTRS, providing a link between CI and NNT in mTRS regulation. We show that (i) mTRS is enhanced by simultaneous activation of CI and complex II (CII); (ii) CI is the principal origin of mTRS; (iii) NNT regulates mTRS via NADH- and ∆Ψmt-dependent mechanisms; (iv) mTRS is not a pH spike; and (v), mTRS changes in amplitude under stress conditions and its occurrence can be a signature of mitochondrial health. Collectively, we uncovered and characterized the biophysical properties and mechanisms of mTRS, and propose it as a potential diagnostic tool for CI-related dysfunctions, and as a biomarker of mitochondrial functional integrity.

Structure of bovine cytochrome c oxidase crystallized at a neutral pH using a fluorinated detergent.

Cytochrome c oxidase (CcO) couples proton pumping to O2 reduction. Its enzymatic activity depends sensitively on pH over a wide range. However, owing to difficulty in crystallizing this protein, X-ray structure analyses of bovine CcO aimed at understanding its reaction mechanism have been conducted using crystals prepared at pH 5.7, which is significantly lower than that in the cell. Here, oxidized CcO at pH 7.3 was crystallized using a fluorinated octyl-maltoside derivative, and the structure was determined at 1.77 Šresolution. No structural differences between crystals obtained at the neutral pH and the acidic pH were detected within the molecules. On the other hand, some differences in intermolecular interactions were detected between the two types of crystal. The influence of pH on the molecular surface is likely to contribute to the pH dependency of the aerobic oxidation of ferrocytochrome c.

Subconductance states of mitochondrial chloride channels: implication for functionally-coupled tetramers.

Recently, it has been discovered that isoforms of intracellular chloride channels (CLIC) are present in cardiac mitochondria. By reconstituting rat cardiac mitochondrial chloride channels into bilayer lipid membranes, we detected three equally separated subconductance states with conductance increment of 45 pS and < 2% occupancy. The observed rare events of channel decomposition into substates, accompanied by disrupted gating, provide an insight into channel quaternary structure. Our findings suggest that the observed channels work as four functionally coupled subunits with synchronized gating. We discuss the putative connection of channel activity from native mitochondria with the recombinant CLIC channels. However, conclusive evidence is needed to prove this connection.

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex III.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex II.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882-9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: (a) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site IIf); (b) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site IIf The decrease in matrix [dicarboxylate] allows O2 access to reduced site IIf, thereby making electron donation to O2 possible, explaining the rapid increase in ROS production provided that site IIf is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased.

Identity and function of a cardiac mitochondrial small conductance Ca2+-activated K+ channel splice variant.

We provide evidence for location and function of a small conductance, Ca2+-activated K+ (SKCa) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SKCa agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SKCa antagonists worsened IR injury. Intravenous infusion of a SKCa channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca2+ calmodulin binding domain is required for Ca2+ sensing to induce mK+ influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK+ influx was driven by adding CaCl2. Moreover, there was a greater fall in membrane potential (ΔΨm), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SKCa channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SKCa channel opening remains unclear. SKCa channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa2+ overload.

Identification and evolutionary analysis of tissue-specific isoforms of mitochondrial complex I subunit NDUFV3.

Mitochondrial complex I is the largest respiratory chain complex. Despite the enormous progress made studying its structure and function in recent years, potential regulatory roles of its accessory subunits remained largely unresolved. Complex I gene NDUFV3, which occurs in metazoa, contains an extra exon that is only present in vertebrates and thereby evolutionary even younger than the rest of the gene. Alternative splicing of this extra exon gives rise to a short NDUFV3-S and a long NDUFV3-L protein isoform. Complexome profiling revealed that the two NDUFV3 isoforms are constituents of the multi-subunit complex I. Further mass spectrometric analyses of complex I from different murine and bovine tissues showed a tissue-specific expression pattern of NDUFV3-S and NDUFV3-L. Hence, NDUFV3-S was identified as the only isoform in heart and skeletal muscle, whereas in liver, brain, and lung NDUFV3-L was expressed as the dominant isoform, together with NDUFV3-S present in all tissues analyzed. Thus, we identified NDUFV3 as the first out of 30 accessory subunits of complex I present in vertebrate- and tissue-specific isoforms. Interestingly, the tissue-specific expression pattern of NDUFV3-S and NDUFV3-L isoforms was paralleled by changes in kinetic parameters, especially the substrate affinity of complex I. This may indicate a regulatory role of the NDUFV3 isoforms in different vertebrate tissues.

Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.