Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples
2021; Elsevier BV; Volume: 297; Issue: 4 Linguagem: Inglês
10.1016/j.jbc.2021.101204
ISSN1083-351X
AutoresFariha Ansari, Belem Yoval‐Sánchez, Zoya Niatsetskaya, Sergey A. Sosunov, Анна Степанова, Christian Garcia, Edward Owusu-Ansah, Vadim S. Ten, Ilka Wittig, Alexander Galkin,
Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoImpairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min−1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies. Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min−1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies. Mitochondrial complex I (Enzyme Commission number: 1.6.5.3) is a key component of the respiratory chain that catalyzes oxidation of matrix NADH by ubiquinone, which is coupled with proton translocation across the inner mitochondrial membrane (see Refs. (1Hirst J. Mitochondrial complex I.Annu. Rev. Biochem. 2013; 82: 551-575Crossref PubMed Scopus (370) Google Scholar, 2Wirth C. Brandt U. Hunte C. Zickermann V. Structure and function of mitochondrial complex I.Biochim. Biophys. Acta. 2016; 1857: 902-914Crossref PubMed Scopus (164) Google Scholar) for reviews). The enzyme contains one molecule of FMN tightly bound to the 51 kDa (NDUFV1) subunit that was first determined biochemically (3Hatefi Y. Rieske J.S. Preparation and properties of DPNH-coenzyme Q reductase (complex I of the respiratory chain).Methods Enzymol. 1967; 10: 235-239Crossref Scopus (123) Google Scholar, 4Ragan C.I. The structure and subunit composition of the particulate NADH-ubiquinone reductase of bovine heart mitochondria.Biochem. J. 1976; 154: 295-305Crossref PubMed Scopus (35) Google Scholar, 5Rao N. Felton S.P. Huennekens F.M. Quantitative determination of mitochondrial flavins.Methods Enzymol. 1967; 10: 494-499Crossref Scopus (24) Google Scholar, 6Belogrudov G. Hatefi Y. Catalytic sector of complex I (NADH:ubiquinone oxidoreductase): Subunit stoichiometry and substrate-induced conformation changes.Biochemistry. 1994; 33: 4571-4576Crossref PubMed Scopus (106) Google Scholar) and then confirmed by structural data (7Sazanov L.A. Hinchliffe P. Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus.Science. 2006; 311: 1430-1436Crossref PubMed Scopus (643) Google Scholar, 8Vinothkumar K.R. Zhu J. Hirst J. Architecture of mammalian respiratory complex I.Nature. 2014; 515: 80-84Crossref PubMed Scopus (286) Google Scholar, 9Fiedorczuk K. Letts J.A. Degliesposti G. Kaszuba K. Skehel M. Sazanov L.A. Atomic structure of the entire mammalian mitochondrial complex I.Nature. 2016; 537: 644-648PubMed Google Scholar). The FMN molecule serves as the primary electron acceptor for NADH and transfers electrons downstream to the chain of FeS clusters. FMN-deficient complex I cannot catalyze reactions of the direct (10Gostimskaya I.S. Grivennikova V.G. Cecchini G. Vinogradov A.D. Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH:ubiquinone oxidoreductase (complex I).FEBS Lett. 2007; 581: 5803-5806Crossref PubMed Scopus (25) Google Scholar, 11Holt P.J. Efremov R.G. Nakamaru-Ogiso E. Sazanov L.A. Reversible FMN dissociation from Escherichia coli respiratory complex I.Biochim. Biophys. Acta. 2016; 1857: 1777-1785Crossref PubMed Scopus (27) Google Scholar) or reverse (12Stepanova A. Kahl A. Konrad C. Ten V. Starkov A.S. Galkin A. Reverse electron transfer results in a loss of flavin from mitochondrial complex I: Potential mechanism for brain ischemia reperfusion injury.J. Cereb. Blood Flow Metab. 2017; 37: 3649-3658Crossref PubMed Scopus (39) Google Scholar) physiological reaction and is also not active in reactive oxygen species (ROS) generation (12Stepanova A. Kahl A. Konrad C. Ten V. Starkov A.S. Galkin A. Reverse electron transfer results in a loss of flavin from mitochondrial complex I: Potential mechanism for brain ischemia reperfusion injury.J. Cereb. Blood Flow Metab. 2017; 37: 3649-3658Crossref PubMed Scopus (39) Google Scholar, 13Stepanova A. Sosunov S. Niatsetskaya Z. Konrad C. Starkov A.A. Manfredi G. Wittig I. Ten V. Galkin A. Redox-dependent loss of flavin by mitochondrial complex I in brain ischemia/reperfusion injury.Antioxid. Redox Signal. 2019; 31: 608-622Crossref PubMed Scopus (25) Google Scholar). Impairments of complex I function at that first redox step are associated with several pathophysiological states and genetic diseases (14Fiedorczuk K. Sazanov L.A. Mammalian mitochondrial complex I structure and disease-causing mutations.Trends Cell Biol. 2018; 28: 835-867Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Several human pathological mutations are localized in the NDUFV1 subunit close to the nucleotide-binding site and most likely can affect FMN redox properties, binding affinity, or stability of the enzyme (15Varghese F. Atcheson E. Bridges H.R. Hirst J. Characterization of clinically identified mutations in NDUFV1, the flavin-binding subunit of respiratory complex I, using a yeast model system.Hum. Mol. Genet. 2015; 24: 6350-6360Crossref PubMed Scopus (30) Google Scholar, 16Borna N.N. Kishita Y. Sakai N. Hamada Y. Kamagata K. Kohda M. Ohtake A. Murayama K. Okazaki Y. Leigh syndrome due to NDUFV1 mutations initially presenting as LBSL.Genes (Basel). 2020; 11: 1325Crossref Scopus (2) Google Scholar, 17Incecik F. Herguner O.M. Besen S. Bozdogan S.T. Mungan N.O. Late-onset Leigh syndrome due to NDUFV1 mutation in a 10-year-old boy initially presenting with ataxia.J. Pediatr. Neurosci. 2018; 13: 205-207Crossref PubMed Google Scholar, 18Srivastava A. Srivastava K.R. Hebbar M. Galada C. Kadavigrere R. Su F. Cao X. Chinnaiyan A.M. Girisha K.M. Shukla A. Bielas S.L. Genetic diversity of NDUFV1-dependent mitochondrial complex I deficiency.Eur. J. Hum. Genet. 2018; 26: 1582-1587Crossref PubMed Scopus (5) Google Scholar). Loss of mitochondrial complex I integrity and activity was found in tissues of patients with Parkinson's disease (19Keeney P.M. Xie J. Capaldi R.A. Bennett Jr., J.P. Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled.J. Neurosci. 2006; 26: 5256-5264Crossref PubMed Scopus (543) Google Scholar, 20Swerdlow R.H. Parks J.K. Miller S.W. Tuttle J.B. Trimmer P.A. Sheehan J.P. Bennett Jr., J.P. Davis R.E. Parker Jr., W.D. Origin and functional consequences of the complex I defect in Parkinson's disease.Ann. Neurol. 1996; 40: 663-671Crossref PubMed Scopus (575) Google Scholar), which plays a role in epileptogenesis (21Ryan K. Backos D.S. Reigan P. Patel M. Post-translational oxidative modification and inactivation of mitochondrial complex I in epileptogenesis.J. Neurosci. 2012; 32: 11250-11258Crossref PubMed Scopus (60) Google Scholar). Recently, we found that mitochondrial impairment during brain ischemia/reperfusion is due to inactivation of complex I via dissociation of FMN from the holoenzyme (13Stepanova A. Sosunov S. Niatsetskaya Z. Konrad C. Starkov A.A. Manfredi G. Wittig I. Ten V. Galkin A. Redox-dependent loss of flavin by mitochondrial complex I in brain ischemia/reperfusion injury.Antioxid. Redox Signal. 2019; 31: 608-622Crossref PubMed Scopus (25) Google Scholar, 22Kahl A. Stepanova A. Konrad C. Anderson C. Manfredi G. Zhou P. Iadecola C. Galkin A. Critical role of flavin and glutathione in complex I-mediated bioenergetic failure in brain ischemia/reperfusion injury.Stroke. 2018; 49: 1223-1231Crossref PubMed Scopus (47) Google Scholar) (see Ref. (23Galkin A. Brain ischemia/reperfusion injury and mitochondrial complex I damage.Biochemistry (Mosc.). 2019; 84: 1411-1423Crossref PubMed Scopus (28) Google Scholar) for review). Loss of flavin from the enzyme was also observed during reperfusion of ischemic liver and kidneys used for transplantation (24Schlegel A. Muller X. Mueller M. Stepanova A. Kron P. de Rougemont O. Muiesan P. Clavien P.A. Galkin A. Meierhofer D. Dutkowski P. Hypothermic oxygenated perfusion protects from mitochondrial injury before liver transplantation.EBioMedicine. 2020; 60: 103014Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 25Wang L. Thompson E. Bates L. Pither T.L. Hosgood S.A. Nicholson M.L. Watson C.J.E. Wilson C. Fisher A.J. Ali S. Dark J.H. Flavin mononucleotide as a biomarker of organ quality-a pilot study.Transplant Direct. 2020; 6e600Crossref PubMed Scopus (15) Google Scholar). Rapidly rising interest in biomedically oriented studies of mitochondrial complex I demands a reliable procedure for fast and accurate determination of enzyme content in a given sample. The method presented here allows quantitative analysis of complex I–bound FMN content and is based on native polyacrylamide gel electrophoresis and fluorimetric scanning. This approach has great potential use for the determination of absolute content and catalytic turnover number of complex I in preparations obtained from genetic mutants, tissues ex vivo, and human biopsies. Our study provides a valuable tool for utilizing complex I content in homogenates and cells as a valid biomarker. A typical Coomassie-stained high-resolution clear native electrophoresis gel is shown in Figure 1A, lane 2. The complex I band was stained by an "in-gel" NADH:nitro tetrazolium reaction catalyzed by the enzyme (Fig. 1A, lane 3). Flavin fluorescent scanning of a gel (excitation of 473 nm/emission of 530 nm) showed bright flavin fluorescence in the upper part of the gel in a band of apparent molecular weight 2.5 to 2.7 MDa (F1 band) (Fig. 1A, lane 5). Flavin of complex I was not detected in the native gel because enzyme-bound FMN fluorescence is quenched in the intact enzyme (26Kunz W.S. Kunz W. Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria.Biochim. Biophys. Acta. 1985; 841: 237-246Crossref PubMed Scopus (104) Google Scholar, 27Hassinen I.E. From identification of fluorescent flavoproteins to mitochondrial redox indicators in intact tissues.J. Innov. Opt. Health Sci. 2014; 07: 1350058Crossref Scopus (6) Google Scholar, 28Blinova K. Levine R.L. Boja E.S. Griffiths G.L. Shi Z.D. Ruddy B. Balaban R.S. Mitochondrial NADH fluorescence is enhanced by complex I binding.Biochemistry. 2008; 47: 9636-9645Crossref PubMed Scopus (77) Google Scholar). However, complex I FMN fluorescence can be revealed after denaturation of the complex I molecule. After the addition of 20% SDS directly on top of the gel, a strong flavin fluorescence signal appeared as a single band corresponding to the position of complex I (∼850 kDa, F2 band). The MS/MS identification of FMN-containing bands F1 and F2 is given in Table 1. MS/MS analysis indicates that band F1 corresponds to the flavin adenine dinucleotide (FAD)–containing dihydrolipoyl dehydrogenase (DLD) subunit of α-ketoglutarate dehydrogenase complex (KGDHC) and band F2 to mitochondrial complex I, which FMN fluorescence revealed only after denaturation of the enzyme. The characteristic fluorescence emission spectrum of flavin was found in extracts from the F2 band (Fig. 1B).Table 1List of most abundant mitochondrial proteins identified by LC–MS/MS in the flavin fluorescent gel bands F1 and F2 resolved by hrCNE from Figure 1NumberProtein nameMolecular weight (kDa)Access number (National Centerfor Biotechnology Information)Protein spectrum countaProtein spectrum count was normalized by the total spectral count. The detailed MS data are provided in Table S1.F1F212-oxoglutarate dehydrogenase118.18Q60597-341562Dihydrolipoyllysine-succinyltransferase48.99Q9D2G227713OGDLH protein114.54B2RXT3_MOUSE23434MICOS complex subunit Mic6082.93Q8CAQ8-2234705ADP/ATP translocase 232.93ADT2_MOUSE90326Dihydrolipoyl dehydrogenase54.27DLDH_MOUSE5355NADH-ubiquinone oxidoreductase 75 kDa79.70Ndufs1333286ATP synthase subunit beta59.75Atp5f1b792427NADH dehydrogenase flavoprotein 149.91Ndufv1181128NADH dehydrogenase FeS protein 252.63Ndufs2201079NADH dehydrogenase subunit 1040.60Ndufa10159310NADH dehydrogenase subunit 942.12Ndufa91671a Protein spectrum count was normalized by the total spectral count. The detailed MS data are provided in Table S1. Open table in a new tab Denaturation-induced FMN fluorescence is not instantaneous and takes at least 15 min for the signal to fully develop for 1-mm gels (Fig. 2A). Longer time exposure resulted in broadening of the band, most likely because of the diffusion of the cofactor through the porous gel. The intensity of the upper band F1 does not change upon SDS treatment. The fluorescent scan of an SDS-treated gel with different amounts of loaded protein is shown in Figure 2B (range of 13–74 μg protein/well). Densitometric analysis of the bands' intensity showed a linear dependence of the band fluorescence on the amount of loaded protein (Fig. 2C). The flavin fluorescence signal from complex I (FMN) or KGDHC bands in gels can be quantitatively estimated by calibration with a known amount of flavin added directly on the gel surface during scanning. The sensitivity of the Typhoon instrument was estimated to be 20 to 40 fmol of FMN, and the fluorescence intensity depended linearly on the amount of FMN added in the range of 0.3 to 3 pmol (Fig. 2D). Based on the calibration, we calculated absolute content of complex I and KGDHC in our brain mitochondria samples. Complex I contains one FMN per molecule of enzyme, therefore the calculated complex I content in intact brain mitochondria was determined as 18.6 ± 0.9 pmol/mg protein (n = 4). FAD fluorescence is only 15% of that of FMN (29Kozioł J. Fluorometric analysis of riboflavin and its coenzymes.Methods Enzymol. 1971; 18 B: 253-285Crossref Scopus (145) Google Scholar, 30Galban J. Sanz-Vicente I. Navarro J. de Marcos S. The intrinsic fluorescence of FAD and its application in analytical chemistry: A review.Methods Appl. Fluoresc. 2016; 4042005Crossref PubMed Scopus (36) Google Scholar), and from our calibration, the calculated content of FAD of DLD (upper F1 band in Fig. 1A) was determined as 65.0 ± 4.7 pmol FAD/mg protein (Table 2).Table 2Analysis of complex I content and catalytic turnover number in whole tissue homogenates from different mouse tissues, intact mouse brain mitochondria, bovine mitochondria, and SMPPreparation typeKGDHC FAD contentpmol/mg proteinComplex I FMN contentpmol/mg proteinNADH:HARaActivities were assayed accordingly to the Experimental procedures section at 25 °C in buffer made of 125 mM KCl, 0.02 mM EGTA, 20 mM Hepes–KOH (pH 7.5) and either 1 mM HAR or 40 to 50 μM Q1. About 100 μM of NADH was added to 2 to 30 or 50 to 400 μg/ml protein for HAR and Q1 reactions, respectively. Values are given in μmol NADH × min−1 × mg protein−1.NADH:Q1aActivities were assayed accordingly to the Experimental procedures section at 25 °C in buffer made of 125 mM KCl, 0.02 mM EGTA, 20 mM Hepes–KOH (pH 7.5) and either 1 mM HAR or 40 to 50 μM Q1. About 100 μM of NADH was added to 2 to 30 or 50 to 400 μg/ml protein for HAR and Q1 reactions, respectively. Values are given in μmol NADH × min−1 × mg protein−1.Activityμmol × min−1 × mg−1TurnoverbEnzyme catalytic turnover (kcat) was calculated as moles of NADH oxidized per minute per mole of enzyme by dividing activity values in μmol × min−1 × mg protein−1 by enzyme content in micromoles of complex I per milligram of protein.×104, min−1Activityμmol × min−1 × mg−1TurnoverbEnzyme catalytic turnover (kcat) was calculated as moles of NADH oxidized per minute per mole of enzyme by dividing activity values in μmol × min−1 × mg protein−1 by enzyme content in micromoles of complex I per milligram of protein.×104, min−1Brain17 ± 110 ± 10.91 ± 0.068.96 ± 1.000.105 ± 0.0041.03 ± 0.10Heart38 ± 850 ± 55.39 ± 0.4010.9 ± 1.290.229 ± 0.0220.46 ± 0.06LiverND6 ± 10.75 ± 0.0312.1 ± 1.150.037 ± 0.0020.60 ± 0.06Kidneys16 ± 826 ± 31.62 ± 0.036.28 ± 0.800.125 ± 0.0050.48 ± 0.06Muscle50 ± 320 ± 11.60 ± 0.107.87 ± 0.710.076 ± 0.0100.37 ± 0.05MB MtcIntact mitochondria from mouse brain (MB Mt), bovine heart (BH Mt), and bovine heart SMP (BH SMP).65 ± 519 ± 11.62 ± 0.048.68 ± 0.470.21 ± 0.021.06 ± 0.03BH MtcIntact mitochondria from mouse brain (MB Mt), bovine heart (BH Mt), and bovine heart SMP (BH SMP).123 ± 951 ± 44.46 ± 0.168.80 ± 0.740.135 ± 0.0070.27 ± 0.02BH SMPcIntact mitochondria from mouse brain (MB Mt), bovine heart (BH Mt), and bovine heart SMP (BH SMP).ND70 ± 65.86 ± 0.128.43 ± 0.800.071 ± 0.0050.10 ± 0.01Abbreviation: ND, not detected.a Activities were assayed accordingly to the Experimental procedures section at 25 °C in buffer made of 125 mM KCl, 0.02 mM EGTA, 20 mM Hepes–KOH (pH 7.5) and either 1 mM HAR or 40 to 50 μM Q1. About 100 μM of NADH was added to 2 to 30 or 50 to 400 μg/ml protein for HAR and Q1 reactions, respectively. Values are given in μmol NADH × min−1 × mg protein−1.b Enzyme catalytic turnover (kcat) was calculated as moles of NADH oxidized per minute per mole of enzyme by dividing activity values in μmol × min−1 × mg protein−1 by enzyme content in micromoles of complex I per milligram of protein.c Intact mitochondria from mouse brain (MB Mt), bovine heart (BH Mt), and bovine heart SMP (BH SMP). Open table in a new tab Abbreviation: ND, not detected. Based on complex I content values, it is possible to determine the enzyme turnover number in NADH-dependent activities of complex I in brain mitochondria. After solubilization of mitochondrial membranes with n-dodecyl-β-d-maltoside (DDM) and separation of supernatant, NADH:hexaammineruthenium chloride (II) (HAR) and NADH:Q1 oxidoreductase activities were measured before applying onto an hrCN gel (Table 2). In the past, we showed that brain mitochondrial complex I can lose its natural cofactor FMN when incubated in conditions of so-called reverse electron transfer (RET) (12Stepanova A. Kahl A. Konrad C. Ten V. Starkov A.S. Galkin A. Reverse electron transfer results in a loss of flavin from mitochondrial complex I: Potential mechanism for brain ischemia reperfusion injury.J. Cereb. Blood Flow Metab. 2017; 37: 3649-3658Crossref PubMed Scopus (39) Google Scholar, 13Stepanova A. Sosunov S. Niatsetskaya Z. Konrad C. Starkov A.A. Manfredi G. Wittig I. Ten V. Galkin A. Redox-dependent loss of flavin by mitochondrial complex I in brain ischemia/reperfusion injury.Antioxid. Redox Signal. 2019; 31: 608-622Crossref PubMed Scopus (25) Google Scholar, 22Kahl A. Stepanova A. Konrad C. Anderson C. Manfredi G. Zhou P. Iadecola C. Galkin A. Critical role of flavin and glutathione in complex I-mediated bioenergetic failure in brain ischemia/reperfusion injury.Stroke. 2018; 49: 1223-1231Crossref PubMed Scopus (47) Google Scholar). In that state, electrons from succinate are directed to ubiquinone, reducing it to ubiquinol, and electrons are then transferred to complexes III and IV and oxygen is reduced to water. This generates a proton-motive force across the inner mitochondrial membrane, and a small fraction of electrons from ubiquinol is pushed upstream reducing all redox centers of complex I. Maintaining complex I FMN in the reduced state results in dissociation of FMNH2 from its binding site (12Stepanova A. Kahl A. Konrad C. Ten V. Starkov A.S. Galkin A. Reverse electron transfer results in a loss of flavin from mitochondrial complex I: Potential mechanism for brain ischemia reperfusion injury.J. Cereb. Blood Flow Metab. 2017; 37: 3649-3658Crossref PubMed Scopus (39) Google Scholar, 13Stepanova A. Sosunov S. Niatsetskaya Z. Konrad C. Starkov A.A. Manfredi G. Wittig I. Ten V. Galkin A. Redox-dependent loss of flavin by mitochondrial complex I in brain ischemia/reperfusion injury.Antioxid. Redox Signal. 2019; 31: 608-622Crossref PubMed Scopus (25) Google Scholar, 22Kahl A. Stepanova A. Konrad C. Anderson C. Manfredi G. Zhou P. Iadecola C. Galkin A. Critical role of flavin and glutathione in complex I-mediated bioenergetic failure in brain ischemia/reperfusion injury.Stroke. 2018; 49: 1223-1231Crossref PubMed Scopus (47) Google Scholar). To validate our method, we tested if this treatment results in a decreased complex I-associated FMN fluorescence signal. A typical flavin fluorescent gel of solubilized brain mitochondria before and after incubation in RET conditions is shown in Figure 3A. About 20 min of incubation in RET-like conditions resulted in a dramatic decrease of the complex I-associated FMN signal but had no effect on FAD from KGDHC. The quantification of various mitochondrial proteins gives insight into the function and role of mitochondria in different tissues. It was of interest to find out whether this approach can be used for the measurements of complex I content and catalytic turnover number in different tissues without isolation of mitochondria first. We developed a fast and an accurate method based on whole tissue homogenate analysis. Flavin fluorescence of a representative gel containing protein solubilized from the brain, heart, liver, muscle, and kidneys homogenate is shown in Figure 4. Knowing the turnover number (kcat, catalytic rate constant) of an enzyme is essential for understanding its regulation and function. By determining complex I content and specific activity, we calculated complex I catalytic turnover number in NADH:HAR and NADH:Q1 reductase reactions of complex I in homogenates from different tissues (Table 2). As expected, the highest complex I content was found in heart tissue, and values were strikingly similar between mouse heart and isolated bovine heart mitochondria. Moreover, while complex I content was different for mouse brain homogenate and isolated brain mitochondria, the turnover values were the same. Bovine heart mitochondria and submitochondrial particles (SMPs) are the classical object of study in bioenergetics; therefore, we determined their complex I content and enzymatic turnover number (Table 2). Among all studied preparations, SMPs have the highest complex I content (70 ± 6 pmol/mg protein). Finally, we validated our method using membrane preparations from different organisms and primary and cultured cells. As shown in Figure 5, SDS treatment of an hrCN gel resulted in the appearance of a single complex I FMN band (apparent molecular weight of 850–1000 kDa) in preparations of bovine, Drosophila, earthworm, and plant mitochondria, as well as primary astrocytes and cultured human embryonic kidney 293 (HEK293) cells. Note that complex I in plants and worms is slightly larger than the mammalian enzyme probably because of the presence of extra subunits in plant complex I (31Heazlewood J.L. Howell K.A. Millar A.H. Mitochondrial complex I from arabidopsis and rice: Orthologs of mammalian and fungal components coupled with plant-specific subunits.Biochim. Biophys. Acta. 2003; 1604: 159-169Crossref PubMed Scopus (158) Google Scholar, 32Maldonado M. Padavannil A. Zhou L. Guo F. Letts J.A. Atomic structure of a mitochondrial complex I intermediate from vascular plants.Elife. 2020; 9e56664Crossref PubMed Google Scholar) or tight association of the enzyme with complex III at a low DDM/protein ratio as was shown for the Caenorhabditis elegans enzyme (33Suthammarak W. Yang Y.Y. Morgan P.G. Sedensky M.M. Complex I function is defective in complex IV-deficient Caenorhabditis elegans.J. Biol. Chem. 2009; 284: 6425-6435Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Bacterial complex I is more fragile than the mitochondrial enzyme (34Berrisford J.M. Baradaran R. Sazanov L.A. Structure of bacterial respiratory complex I.Biochim. Biophys. Acta. 2016; 1857: 892-901Crossref PubMed Scopus (50) Google Scholar), and in our hands, separation of Escherichia coli membranes resulted in two complex I flavin bands of around 550 and 170 kDa (also displaying in-gel activity, not shown). The low molecular weight band corresponds to the NADH dehydrogenase module of E. coli complex I (subunits NuoE, F, and G; total molecular weight of 170 kDa) and was observed previously after detergent treatment complex I preparations at pH >6 (35Leif H. Sled V.D. Ohnishi T. Weiss H. Friedrich T. Isolation and characterization of the proton-translocating NADH:ubiquinone oxidoreductase from Escherichia coli.Eur. J. Biochem. 1995; 230: 538-548Crossref PubMed Scopus (246) Google Scholar) or as an assembly intermediate (36Erhardt H. Steimle S. Muders V. Pohl T. Walter J. Friedrich T. Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli.Biochim. Biophys. Acta. 2012; 1817: 863-871Crossref PubMed Scopus (32) Google Scholar). Cultured cells are an established pipeline for studying the assembly of complex I and clinically relevant mutations (37Stroud D.A. Surgenor E.E. Formosa L.E. Reljic B. Frazier A.E. Dibley M.G. Osellame L.D. Stait T. Beilharz T.H. Thorburn D.R. Salim A. Ryan M.T. Accessory subunits are integral for assembly and function of human mitochondrial complex I.Nature. 2016; 538: 123-126Crossref PubMed Scopus (219) Google Scholar, 38Vogel R.O. Dieteren C.E. van den Heuvel L.P. Willems P.H. Smeitink J.A. Koopman W.J. Nijtmans L.G. Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits.J. Biol. Chem. 2007; 282: 7582-7590Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 39Fernandez-Vizarra E. Lopez-Calcerrada S. Formosa L.E. Perez-Perez R. Ding S. Fearnley I.M. Arenas J. Martin M.A. Zeviani M. Ryan M.T. Ugalde C. SILAC-based complexome profiling dissects the structural organization of the human respiratory supercomplexes in SCAFI(KO) cells.Biochim. Biophys. Acta Bioenerg. 2021; 1862: 148414Crossref PubMed Scopus (8) Google Scholar, 40Perez-Perez R. Lobo-Jarne T. Milenkovic D. Mourier A. Bratic A. Garcia-Bartolome A. Fernandez-Vizarra E. Cadenas S. Delmiro A. Garcia-Consuegra I. Arenas J. Martin M.A. Larsson N.G. Ugalde C. COX7A2L is a mitochondrial complex III binding protein that stabilizes the III2+IV supercomplex without affecting respirasome formation.Cell Rep. 2016; 16: 2387-2398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar); therefore, we tested the versatility of our approach using the HEK293 cell line (Fig. 5B). Harvested cells were homogenized, solubilized using DDM, and the resulting lysate was either applied directly on an hrCN gel or centrifuged, and only the supernatant was loaded in accordance to the original protocol. For both approaches, we obtained very close values of FMN or FAD content. For the DDM-solubilized total cell suspension (no centrifugation), the values of 0.152 ± 0.003 pmol FMN/106 cells and 1.00 ± 0.08 pmol FAD/106 cells were determined for complex I and KGDHC, respectively. Two main strategies are most commonly used when the absolute content of a membrane enzyme such as complex I is to be measured. One is to carefully titrate the activity of the enzyme with a specific tightly bound inhibitor, where complex I content is determined as the intersection points from the linear graphs of the residual activity versus the amount of inhibitor added (per milligram of protein). The main assumption for this method is 1:1 stoichiometry of in
Referência(s)