Identification and Characterization of a Common Set of Complex I Assembly Intermediates in Mitochondria from Patients with Complex I Deficiency

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly. Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly. NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3) is the largest and the least understood of all the respiratory chain complexes. Mammalian complex I is composed of at least 46 subunits, which are encoded by both nuclear (39 subunits) and mitochondrial DNA (7 subunits) (1Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (686) Google Scholar, 2Skehel J.M. Fearnley I.M. Walker J.E. FEBS Lett. 1998; 438: 301-305Crossref PubMed Scopus (59) Google Scholar, 3Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Mol. Cell. Proteomics. 2003; 2: 117-126Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). The complex, which has an estimated molecular mass of almost 1 MDa, catalyzes the transfer of two electrons from NADH to ubiquinone coupled to translocation of four protons across the inner mitochondrial membrane. Low resolution structures based on the electron microscopy of the bovine (4Grigorieff N. J. Mol. Biol. 1998; 277: 1033-1046Crossref PubMed Scopus (304) Google Scholar), Escherichia coli (5Guenebaut V. Schlitt A. Weiss H. Leonard K. Friedrich T. J. Mol. Biol. 1998; 276: 105-112Crossref PubMed Scopus (204) Google Scholar, 6Sazanov L.A. Carroll J. Holt P. Toime L. Fearnley I.M. J. Biol. Chem. 2003; 278: 19483-19491Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and Neurospora crassa (7Guenebaut V. Vincentelli R. Mills D. Weiss H. Leonard K.R. J. Mol. Biol. 1997; 265: 409-418Crossref PubMed Scopus (133) Google Scholar) complex I show that the complex has an L-shaped form with one arm in the membrane and a peripheral arm protruding into the mitochondrial matrix. A second, horseshoe-shaped conformation of the E. coli complex I has also recently been proposed (8Bottcher B. Scheide D. Hesterberg M. Nagel-Steger L. Friedrich T. J. Biol. Chem. 2002; 277: 17970-17977Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The complex can be dissociated by treatment with detergent into three subcomplexes: Iα, corresponding to the peripheral arm and composed of ∼21 mostly hydrophilic subunits, and Iβ and Iγ, both of which make up the membrane arm (9Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar). Subcomplex Iα contains the NADH binding site and most of the redox centers. All of the mtDNA-encoded 1The abbreviations used are: mtDNA, mitochondrial DNA; BN-PAGE, Blue-Native PAGE; COX, cytochrome c oxidase; Alu, restriction endonuclease; Alu-FISH, Alu-PCR repeats fluorescence in situ hybridization; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. subunits and at least 16 of the nuclear-encoded subunits are found in the Iβ and Iγ fractions of the membrane arm. Deficiencies in complex I are among the most common respiratory chain defects (10Wallace D.C. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2655) Google Scholar, 11Loeffen J. Elpeleg O. Smeitink J. Smeets R. Stockler-Ipsiroglu S. Mandel H. Sengers R. Trijbels F. van den Heuvel L. Ann. Neurol. 2001; 49: 195-201Crossref PubMed Scopus (165) Google Scholar, 12van den Heuvel L. Ruitenbeek W. Smeets R. Gelman-Kohan Z. Elpeleg O. Loeffen J. Trijbels F. Mariman E. de Bruijn D. Smeitink J. Am. J. Hum. Genet. 1998; 62: 262-268Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 13Triepels R. Smeitink J. Loeffen J. Smeets R. Trijbels F. van den Heuvel L. Hum. Genet. 2000; 106: 385-391Crossref PubMed Scopus (15) Google Scholar, 14Loeffen J. Smeitink J. Triepels R. Smeets R. Schuelke M. Sengers R. Trijbels F. Hamel B. Mullaart R. van den Heuvel L. Am. J. Hum. Genet. 1998; 63: 1598-1608Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 15Schuelke M. Smeitink J. Mariman E. Loeffen J. Plecko B. Trijbels F. Stockler-Ipsiroglu S. van den Heuvel L. Nat. Genet. 1999; 21: 260-261Crossref PubMed Scopus (243) Google Scholar). Mutations in the mtDNA-encoded subunits of complex I were the first to be associated with a respiratory chain disorder, Lebers hereditary optic neuropathy (10Wallace D.C. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2655) Google Scholar, 16Wallace D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8739-8746Crossref PubMed Scopus (446) Google Scholar). Lebers hereditary optic neuropathy presents with the comparatively mild phenotype of adult-onset blindness due to optic nerve degeneration, and most cases are caused by mutations in one of four complex I subunit genes (MTND1, MTND4, MTND5, and MTND6) (17Wallace D.C. Singh G. Lott M.T. Hodge J.A. Schurr T.G. Lezza A.M. Elsas L.J. Nikoskelainen E.K. Science. 1988; 242: 1427-1430Crossref PubMed Scopus (2066) Google Scholar, 18Man P.Y. Turnbull D.M. Chinnery P.F. J. Med. Genet. 2002; 39: 162-169Crossref PubMed Scopus (387) Google Scholar). In contrast, the majority of early onset complex I deficiencies are severe, and often fatal, autosomal recessive disorders. DNA sequence analysis in more than 20 complex I patients has revealed mutations in seven structural genes: NDUFS2 (11Loeffen J. Elpeleg O. Smeitink J. Smeets R. Stockler-Ipsiroglu S. Mandel H. Sengers R. Trijbels F. van den Heuvel L. Ann. Neurol. 2001; 49: 195-201Crossref PubMed Scopus (165) Google Scholar); NDUFS4 (12van den Heuvel L. Ruitenbeek W. Smeets R. Gelman-Kohan Z. Elpeleg O. Loeffen J. Trijbels F. Mariman E. de Bruijn D. Smeitink J. Am. J. Hum. Genet. 1998; 62: 262-268Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 19Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (121) Google Scholar, 20Budde S.M. van den Heuvel L.P. Janssen A.J. Smeets R.J. Buskens C.A. DeMeirleir L. Van Coster R. Baethmann M. Voit T. Trijbels J.M. Smeitink J.A. Biochem. Biophys. Res. Commun. 2000; 275: 63-68Crossref PubMed Scopus (169) Google Scholar); NDUFS7 (13Triepels R. Smeitink J. Loeffen J. Smeets R. Trijbels F. van den Heuvel L. Hum. Genet. 2000; 106: 385-391Crossref PubMed Scopus (15) Google Scholar); NDUFS8 (14Loeffen J. Smeitink J. Triepels R. Smeets R. Schuelke M. Sengers R. Trijbels F. Hamel B. Mullaart R. van den Heuvel L. Am. J. Hum. Genet. 1998; 63: 1598-1608Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar); NDUFV1 (15Schuelke M. Smeitink J. Mariman E. Loeffen J. Plecko B. Trijbels F. Stockler-Ipsiroglu S. van den Heuvel L. Nat. Genet. 1999; 21: 260-261Crossref PubMed Scopus (243) Google Scholar, 21Benit P. Chretien D. Kadhom N. de Lonlay-Debeney P. Cormier-Daire V. Cabral A. Peudenier S. Rustin P. Munnich A. Rotig A. Am. J. Hum. Genet. 2001; 68: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar); NDUFS1 (21Benit P. Chretien D. Kadhom N. de Lonlay-Debeney P. Cormier-Daire V. Cabral A. Peudenier S. Rustin P. Munnich A. Rotig A. Am. J. Hum. Genet. 2001; 68: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar); and NDUFV2 (22Benit P. Beugnot R. Chretien D. Giurgea I. De Lonlay-Debeney P. Issartel J.P. Corral-Debrinski M. Kerscher S. Rustin P. Rotig A. Munnich A. Hum. Mutat. 2003; 21: 582-586Crossref PubMed Scopus (144) Google Scholar). Structural gene mutations were not found in about 50% of patients in these studies, suggesting that genes coding for assembly factors may be important causes of complex I deficiency. An assembly defect in complex I was described in a patient with a mutation in NDUFS4 (19Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (121) Google Scholar) and in a patient with a mutation in the mitochondrially encoded subunit ND4 (23Hofhaus G. Attardi G. EMBO J. 1993; 12: 3043-3048Crossref PubMed Scopus (88) Google Scholar). Saccharomyces cerevisiae is widely used as a model organism for mitochondrial respiratory chain function but does not contain a complex I (24Buschges R. Bahrenberg G. Zimmermann M. Wolf K. Yeast. 1994; 10: 475-479Crossref PubMed Scopus (28) Google Scholar, 25Nosek J. Fukuhara H. J. Bacteriol. 1994; 176: 5622-5630Crossref PubMed Google Scholar). The current model of assembly of complex I is based on pulse-chase labeling of assembly intermediates in N. crassa and on the characterization of assembly subcomplexes in N. crassa mutants (26Nehls U. Friedrich T. Schmiede A. Ohnishi T. Weiss H. J. Mol. Biol. 1992; 227: 1032-1042Crossref PubMed Scopus (76) Google Scholar, 27Tuschen G. Sackmann U. Nehls U. Haiker H. Buse G. Weiss H. J. Mol. Biol. 1990; PubMed Scopus Google Scholar, R. A. Schmiede A. U. J. Mol. Biol. 1998; 283: PubMed Scopus Google Scholar, F. M. A.M. A. Biochem. J. 1999; PubMed Google Scholar, M. R. A. PubMed Google Scholar); however, of complex I assembly is the of complex I subcomplexes in patients with complex I deficiency due to mutations in either nuclear or mitochondrial DNA and in a human/mouse hybrid cell line with a severe complex I deficiency due to an almost complete to assemble the holoenzyme complex. Blue-Native and a panel of antibodies for structural subunits of complex identified a common of complex I and propose a possible pathway for complex I assembly which differs significantly from that proposed for of the of patient was a in M. J. N. J. Med. 2002; PubMed Scopus Google Scholar) from and were and was and a were The was associated with severe and a muscle revealed the of analysis of the muscle revealed a complex I deficiency of The analysis of a in the gene of in the patient muscle the a which a the of patient was a with a of severe associated with and of patient muscle revealed a in complex I activity of respiratory chain were analysis revealed mitochondrial with The also from a and severe from both complex I activity of from both of from both with the complex I that the defect in patient and is of nuclear and are and at the of both from mitochondrial and and at has a of onset and has a in both patients, with and Complex I activity in muscle mitochondria from patient was and from patient was of the All respiratory chain enzyme were analysis of from patient muscle of a and of all the genes and all the mtDNA-encoded complex I genes Low complex I activity was also found in from patient and all respiratory chain enzyme were of patients with the complex I that the defect in patients and is of nuclear of from patient with from patient complex I that to the as was from all patients, and were by the were from and by with the 16 human gene and the catalytic of human Holt M. Nat. Genet. 1999; 21: PubMed Scopus Google Scholar). The were in and bovine The cell line of was in bovine and were with a to 1993; PubMed Scopus Google Scholar). were with and 16 The were in 1 and for of from patients and and were and at in 20 were for at to were by for 20 at was by the Biochem. PubMed Scopus Google Scholar). were by treatment with or of of as described P. Van den J. Biochem. PubMed Scopus Google Scholar). or mitochondria were with and of was used for H. G. Biochem. PubMed Scopus Google Scholar) was used for of in the first dimension on or for complex I and cytochrome c activity were as described E. L. F. 1997; PubMed Scopus Google Scholar). the two-dimensional of the first dimension gel were for in and and was used to the in the second dimension H. G. Biochem. PubMed Scopus Google Scholar). were detected by analysis and and complex I antibodies against subunits and were from antibodies against complex I subunits were by R. and B. and J. Walker A. and V. I subunits described in of the nuclear-encoded subunits used in are based on the molecular Iα and membrane arm Iα and Iγ Iα Iα Iα Iα Iβ and membrane arm Iα and Iγ Iα Iα Iγ The of the nuclear-encoded subunits used in are based on the molecular in a of Complex I analysis of mitochondria from muscle of patients and activity of complex the activity of was analysis of COX, complex and complex however, complex I was in all four patients antibodies against both nuclear (39 and and mitochondrial subunits of complex I identified common subcomplexes with molecular of and An of about was in patients and Although the pattern of the subcomplexes was similar in all four patients, the of varied among the The the in and the subunits in patients and was than in patients and in the of and subunits in the subcomplexes was similar or than in the of complex I molecular pattern of the subunit as a and could not be to molecular pattern of the subunit as a and could not be to in a of Complex I the subunit of the complex I used a of 11 antibodies directed against structural subunits of complex I in with two-dimensional mitochondria of from patient were in the first dimension and in the second dimension. of subunits in the complex I from the patient with subcomplexes were present in patient mitochondria In to the five subcomplexes described in the gels subcomplexes of molecular mass and were identified A similar pattern was in muscle mitochondria from patients and not the The of patient mitochondria all subunits and and In all patients, antibodies against subunits and detected these subunits in all the subcomplexes for A similar pattern was found for subunit however, more as a than A second pattern was antibodies to subunits and which were present in subcomplexes and The subunit and also a similar pattern and were not present in the molecular mass Although the does subcomplexes in the the the enzyme and the subcomplexes is than in the The and subunits were detected in the the to patients and and the subunit a that could not be to of the of complex I were also detected in from patient not however, the were in with in muscle In a could be detected with antibodies against the and subunits however, the subcomplexes in the patients were not observed of the and subunit of complex I subcomplexes in patients and mitochondria from patient and patient of were by two-dimensional and the of complex I subunits in the subcomplexes was by The line the of complex and the subcomplexes The patient of is with a The of molecular mass is of Complex I in a to a respiratory chain defect to nuclear or mitochondrial DNA is to patient with to for of the phenotype in a recessive nuclear gene nuclear genes could human nuclear respiratory chain gene in patient cell but observed In of a human fibroblast line with in and complex I analysis a of the on complex I complex was phenotype does not from a of human as analysis a on not to result from the of genes present in the human nuclear and mitochondrial analysis of the the of complex I subcomplexes with molecular of and corresponding to subcomplexes complex could be detected in these and the subunit pattern in the complex I subcomplexes was similar to that found in complex I Blue-Native is a for the analysis of respiratory chain and for and respiratory chain Holt 2002; Scholar). labeling of mitochondrial with with BN-PAGE, the of three intermediates in the assembly of in D. Van den J. Biochem. 1998; PubMed Scopus Google Scholar). with caused by mutations in show a early assembly defect V. M. S. K. L. P. L. G. Zeviani M. T. Ann. Neurol. 1999; PubMed Scopus Google Scholar) in which assembly is the of subunit into the catalytic in the of two early assembly intermediates and patients van den Heuvel L.P. E. I. Trijbels L.A. Smeitink J.A. Biochem. Biophys. Res. Commun. 1999; 265: PubMed Scopus Google Scholar). has also been used to the assembly of a of the complex in the mitochondrial membrane A.J. J. M. M. M.T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). have used and a panel of structural subunit antibodies to complex I subcomplexes in patients with either nuclear or complex I defects. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I with different suggests that the subcomplexes intermediates in the assembly of the holoenzyme complex. against the and subunits of complex I identified five distinct subcomplexes and in the in muscle mitochondria from the four patients studied, and two-dimensional revealed two molecular mass subcomplexes and the respiratory chain conformation on the of the subunits not be to the and the inability to these two subcomplexes on the Although the of the subcomplexes varied among the patients, a pattern was In complex I subunits could be into four and all of the Iα structural described by (9Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar), were present in all The subunit is also found in the Iγ that is found on the of the complex at the the peripheral and the membrane The of the pattern of the subunit in the second dimension gel suggests a to the subunits in the and which form of the peripheral arm Iα were present in subcomplexes and Subcomplex with a molecular mass of not contain of the subunits and a similar among the to different of the holoenzyme complex, the membrane and peripheral were both detected in subcomplexes and the subunit was also found in The contains the and subunits, which are of The described as to the membrane Iβ (9Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar), as a on the and was the subunit that were not to to The of about observed in mitochondria from patients and was not found in or muscle from complex I patients not the of and subunits in the with similar to found in the complex, suggesting a assembly defect in patients and Triepels van den Heuvel L.P. L. Smeitink J.A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) complex I subcomplexes in patients with complex I deficiency a analysis to the of structural subunits subunits and in or and to investigate assembly the subunits into three on and complex I activity in the of and subunits most the of of and subunits were and of and subunits were than from the activity In also and subunits and and subunits into two based on in complex I however, the and subunits to different in have on the of mtDNA-encoded subunits of complex I on enzyme assembly. The of human ND4 subunit caused a of mtDNA-encoded subunits to however, the nuclear subunits in the redox to form an with oxidoreductase activity (23Hofhaus G. Attardi G. EMBO J. 1993; 12: 3043-3048Crossref PubMed Scopus (88) Google Scholar). A similar result has been in an ND4 in a with NADH activity was J. 1999; Scopus Google Scholar). subunits and are for the membrane arm assembly in N. crassa A. Biochem. Biol. 1998; PubMed Scopus Google Scholar), and the is for the membrane arm assembly in mitochondria Attardi G. EMBO J. 1998; PubMed Scopus Google Scholar). In contrast, a lack of the subunit does not complex I assembly in G. Attardi G. Mol. Cell. Biol. PubMed Google Scholar). In the the of the or subunit the assembly of complex the of the ND4 or to the of a with NADH activity P. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). The of the in patient the assembly of complex I and to the of complex I however, NADH activity could not be detected in these subcomplexes by an activity a for the subunit in the assembly of complex I. The of the subunit in the of Iγ in the of the (9Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar) suggests that the of subunit causes the of the complex in patient is about the of nuclear-encoded subunits of complex I in the assembly of the complex. A patient with a mutation in the NDUFS4 gene the subunit was to assemble complex I (19Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (121) Google Scholar), but subcomplexes were described in that the NDUFS4 in N. was the was to assemble an almost of and the and subunits, van den Heuvel L.P. L. Smeitink J.A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), were described in a patient to have a mutation in a complex I assembly The current model of complex I and assembly is based on in N. which contains about subunits, at least three of which are not found in complex I and function remains A. M. Biophys. 2002; PubMed Scopus Google Scholar, J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The and characterization of complex I subcomplexes in to propose a model for complex I assembly in that differs significantly from that proposed for N. assembly intermediates have been identified in the N. crassa the peripheral a and a of the membrane and the membrane arm U. J. 2001; PubMed Scopus Google Scholar). The complex is to assemble by of these of complex I structural subunits holoenzyme in the of one or more of these enzyme subcomplexes A. M. J. 2001; PubMed Scopus Google Scholar); however, mutations in subunits to one arm not with assembly of the arm. the to the of the subcomplexes of the of the peripheral arm have not been have subcomplexes the peripheral arm and of the membrane arm. The of on the human enzyme in to more assembly intermediates are subcomplexes of the peripheral arm and subcomplexes of both suggesting that the peripheral and membrane are not in In the peripheral arm of complex I is as two I and Subcomplex I with subunits, which is in to that are of the membrane subunit is a membrane can that subunit is into the membrane to with the which contains of the peripheral arm and of the membrane is subunits are to in subcomplexes and The of assembly of of the peripheral and and of subunits, in the of The subunits are associated with and produce a complex. and an in the assembly of the membrane arm of the N. crassa complex with the of the membrane arm and with the to form the membrane arm R. A. Schmiede A. U. J. Mol. Biol. 1998; 283: PubMed Scopus Google Scholar). A human of one of has been described R. Smeitink J. Smeets R. van Heuvel L. Hum. Genet. 2002; PubMed Scopus Google Scholar), but function is not and DNA sequence analysis of complex I patients assembly has not revealed mutations in gene R. Smeitink J. Smeets R. van Heuvel L. Hum. Genet. 2002; PubMed Scopus Google Scholar). We the of N. R. M. R. and to the of patients and We M. for the analysis and T. for

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