Complex I & Superoxide:
Production and Responses
It's a Matter of Life and Death
October 14, 2008
Edited by Roderick Capaldi, D.Phil.
Past issues are available for review in the archives.
Table of Contents
I. INTRODUCTION
II. WHAT SITES AND WHICH REACTIONS GENERATE SUPEROXIDE FROM COMPLEX I
III. THE CYSTEINE MODIFICATION QUANDRY: RELEVANCE TO SUPEROXIDE PRODUCTION AS WELL AS PROTECTION FROM THIS SPECIES IN COMPLEX I
IV. COMPLEX I AND APOPTOSIS: THE EMERGING CONNECTION AND LINK TO SUPEROXIDE PRODUCTION
V. ASSEMBLY OF COMPLEX I: AN UPDATE
I. INTRODUCTION
It used to be that Complex I studies were the domain of a relatively few groups characterized by special skills in protein purification, the unusual brain function to keep tabs on an ever increasing number of subunits (now settled at 45), and the remarkable optimism to believe that the complex was interesting. Thanks to colleagues such as Hatefi, Yagi, Walker and Brandt to name a few, we mortals can now participate in the study of, and bask in the realization of, the critical importance of this complex in health and disease.
II. WHAT SITES AND WHICH REACTIONS GENERATE SUPEROXIDE FROM COMPLEX I
Complex I is a predominant site of superoxide production in cells, but in what reaction(s) of Complex I is the superoxide produced? Given the variety of inhibitors of the complex by which to isolate different partial reactions, the site should be simple to determine. Unfortunately this is not proving to be the case because of the variety of controls in place to modulate superoxide production by Complex I, and the multiple electron transfer routes (forward and backward reactions) that are employed under different physiological conditions of cellular respiration.
As pointed out in a recent article by Lambert et al., the rate of superoxide production from Complex I varies greatly depending on the conditions of the measurement. In isolated de-energized complex, the dominant site is (apparently) the flavin. However, in intact mitochondria according to these authors, there are three modes of superoxide production by Complex I. These include 1) during forward electron transfer when the superoxide production is low and 2) when the quinone site is inhibited e.g. by rotenone when it is moderately fast. In both of these conditions superoxide is generated at the flavin. The third route to producing superoxide according to Lambert et al., and others, is via reverse electron transfer from reduced quinone (reduced via Complex II). This is a fast rate which is sensitive to pH gradient, and according to several studies, the superoxide production is from the quinone site or close by, connected to this site by conformational changes.
Lambert et al. set out to evaluate the relative contribution of the different pathways to superoxide production using dipheyleneiodonium, a compound that can form a covalent adduct and block electron transfer in flavins. They found that this reagent inhibited superoxide production in the reverse but not the forward direction of electron transfer by a mechanism other than flavin reaction. While this study does not greatly clarify the mode of superoxide production it does provide a compound that is useful in studying the superoxide production and in possibly modulating it under physiological conditions.
LAMBERT. AJ, BUCKINGHAM. JA, BOYSON. HM & BRAND. MD
Diphenyleneiodenium acultely inhibits reactive oxygen species production by mitochondrial Complex I during reverse but not forward electron transport.
Biochim. Biophys. Acta. (2008) 1777.397-403
Two other recent papers address the site of production of superoxide in Complex I. Kudin et al. claim evidence that flavin is the major donor while Esterhazy et al. provide evidence in favor of non heme iron center N1a as the major producer of this oxidative species.
KUDIN. AP, MALINSKA.D & KUNZ.WS
Sites of generation of reactive oxygen species in homogenates of brain tissue determined with the use of respiratory substrates and inhibitors.
Biocim Biophys.Acta. (2008) 1777. 689-95
ESTERHAZY.D, KING.MS, YAKOVLEV.G & HIRST,J
Production of reactive oxygen species by complex I (NADH:ubiquinone oxidoreductase) from Escherichia coli and comparison to the enzyme from mitochondria.
Biochemistry 47. (2008) 3964-71
Clearly the issue of where and how superoxide is produced by Complex I is not yet decided.
III. THE CYSTEINE MODIFICATION QUANDRY: RELEVANCE TO SUPEROXIDE PRODUCTION AS WELL AS PROTECTION FROM THIS SPECIES IN COMPLEX I
Complex I contains a large number of cysteine residues, many associated with non-heme iron centers but others on the surface of the complex and prime targets for modification. Two modifications in particular have been studied in some detail: S –nitrosation and glutathionylation. For updated information on S-nitrosation, the reader is referred to an excellent recent review by Darley Usmar and colleagues.
HILL. BG & DARLEY-USMAR, VM.
S-nitrosation and thiol switching in the mitochondrion: a new paradigm for cardioprotection in ischemic preconditioning.
Biochem. J. 412 e11-13 (2008).
In a recent study Hurd et al. have extended earlier work from their laboratory examining the interaction between Cys on Complex I and the mitochondrial glutathione pool. Previously they had used isolated Complex I and found glutathione modification of Cys in subunits NDUFS1 (75Kd) and NDUFV1 (51K subunit). In their new study the researchers examine Cys modification of Complex I in oxidatively stressed mammalian mitochondria. They see modification of the 75Kd subunit but not the flavin-containing subunit, which they now explain by exposure of Cys in the 51K subunit only in partly denatured, isolated enzyme. They identify the sites of modification on the 75Kd subunit as Cys 531 and cys 704 and further, show that these Cys are similarly modified by physiologically generated superoxide production.
HURD T.R. ET AL.
Complex I within oxidatively stressed bovine heart mitochondria is glutathionylated on Cys 531 and Cys 704 of the 75KDa subunit.
J. Biol. Chem. 283. 24801-15 (2008).
A new picture of modification of the Cys in the 51KDa subunit has been published very recently. This shows intramolecular disulfide formation involving Cys 125,142,187 and 206 in this subunit using isolated Complex I. Is this real or a result of altered protein state after isolation? Time will tell.
ZHANG L. et al.
Mass spectrometry profiles of superoxide induced intramolecular disulfide in the FMN binding subunit of mitochondrial Complex I
J.Am.Soc. Mass Spectrom. Aug 12 in press (2008).
One important point to be made at this juncture is that Complex I exists physiologically, and as isolated, in (at least) two conformations that are reversible through an active de-active transition. The extent to which modifications described above are related to these conformations needs careful study.
Recent work has added to our understanding of the active/deactive enzyme transition of Complex I. Vinogradov, Brandt and colleagues have shown that a Cys of ND3 (Cys39) becomes accessible in the de-active form but is not available in the active form. They hypothesize that this site may be the one modified by S-nitrosation under pathological conditions during hypoxia.
GALKIN. A. et al.
Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory Complex I.
J.Biol.Chem. 283. 20907-13 (2008).
IV. COMPLEX I AND APOPTOSIS: THE EMERGING CONNECTION AND LINK TO SUPEROXIDE PRODUCTION
Several different lines of evidence link Complex I to apoptosis. The first relates to the protein composition of the Complex. It has been known for some time that Grim 19, a subunit of the complex, is an apoptotic factor. There is another pool of this protein in the cytosol, and so whether it is the organelle portion of Complex I or the cytosolic portion, or some equilibrium of the two fractions that regulates cell death remains to be clarified. In a recent study Lu and Cao establish that Grim19 is essential for maintaining the mitochondrial membrane potential. They do this by dissecting the role of different segments of the protein. Unfortunately the results do not establish which pool of the protein is involved.
LU.H & CAO.X.
Grim 19 is essential for maintainance of mitochondrial membrane potential.
(2008) Mol Biol of the Cell 19. 1893-1902
A second apoptotic factor, Apoptosis Inducing Factor (AIF) interacts with and is important for the assembly of Complex I. The relationship between assembly factor function and apoptotic signaling remains to be clarified.
Additionally there are 3 examples of cleavage of Complex I by various proteases that reduce electron transfer and increase superoxide production to induce apoptosis. Thus Martinvalet et al. showed that the protease Granzyme A is able to enter mitochondria and cleave Complex I to induce apoptosis. The entry of the protease does not perturb normal mitochondrial functioning until there is a build up of ROS due to the inhibition of Complex I. These workers identified the site of Complex I cleavage as NDUFS3.
MARTINVALET.D., DYKXHOORN. DM., FERRINI.R & LIEBERMAN J.
Granzyme A cleaves a mitochondrial complex I protein to initiate caspase-independent cell death.
CELL 133. 681-92 (2008)
Other examples of cleavage of Complex I as a preliminary to or in apoptosis are...
...the caspase 3 cleavage of NDUFS1
RICCI. JE., MUNOZ-PINEDO.C.,FITZGERALD.P., BAILLY-MAITRE.B., PERKINDS.GA., YADAVA.N., SCHEFFLER. IE., ELLISMAN. MH & GREEN.DR
Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain.
Cell 117, 773-86 (2004)
...and cleavage of NDUFV2 and of NDUFB8 by calpain10
ARRINGTON.DD., VAN VLEET.TR & SCHNELLMANN.RG.
Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction.
AM J Physiol. Cell. Physiol 291. C1159-71 (2006)
Other modifications of Complex I occur that affect activity and could possibly be involved in induction of or protection against apoptosis, including the effects on Cys residues described above. In a recent study Finkel and colleagues have provided the first definitive evidence of a control of Complex I activity by acetylation/deacetylation involving Sirtuin 3. They find that in the absence of Sirt3 (using Sirt3-/- mouse embryonic fibroblasts) multiple subunits of Complex I are acetylated and the enzyme activity is reduced. Incubation with exogenous Sirt 3 activiates the complex. Further they find an interaction between Sirt 3 and NDUFA9.
AHN. B-H et al.
A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis.
Proc Natl Acad Sci USA early edition (2008)
V. ASSEMBLY OF COMPLEX I: AN UPDATE
In addition to new insights in understanding the role of Complex I in control of energy metabolism, and in inducing apoptosis, there has been significant progress toward understanding the assembly of this complex. Here are two useful recent reviews of this topic:
LAZAROU.M., THORNBURN.DR. RYAN. MT & McKENZIE M.
Assembly of mitochondrial complex I and defects in disease.
(2008) Biochim. Biophys. Acta May 4 ahead of print
VOGEL.RO. SMEITINK.JAM & NIJTMANS. LGJ
Human mitochondrial complex I assembly: a dynamic and versatile process.
(2007) Biochim Biophys Acta. 1767. 1215-27
Among recently identified factors involved in Complex I assembly is C6OR66, which was identified from patients with observed Complex I deficiency:
SAADA. A. et al.
C6ORF66 is an assembly factor of mitochondrial Complex I
(2008) Am. J. Hum. Genet 82. 32-8
...and Oxa1l;
STIBUREK.L. et al.
Knockdown of human Oxa 1l impairs the biogenesis of F1F0 ATP synthase and NADH ubiquinone oxidoreductase
(2007) J. Mol. Biol. 374. 506-16.
...and C8orf38 (this protein was identified in a systems biology approach search of proteins important for Complex I structure and functioning);
PAGLIARINI. DJ. Et al.
A mitochondrial protein compendium elucidates Complex I disease biology
(2008) Cell. 134. 112-23.
Almost half of patients with Complex I deficiency have no identified mutation in Complex I subunits. Assembly of the Complex must include many steps to produce the membrane intercalated and peripheral part of the complex while intercalating the flavin and multiple non-heme iron centers. Thus, the work of Pagliarini et al. not withstanding, it is likely that there are more assembly factors of Complex I to be discovered.
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Recent Research Using MitoSciences' Products for Complex I
A. MS101 for immunocapture of the complex for evaluation of subunit composition, post-translational modifications, etc.1. Ahn B-H et al.Proc Natl Acad Sci USA 105. 14447-52 (2008) 2. Davis MP. et al. J Anim. Sci 86 E-suppl 2/J 60 (2008) 3. Qi X. et al. Inv. Opthalmology and Visual Sci 48. 1-13 (2007) 4. Devi L. et al. J. Biol. Chem 283 9089-97 (2008) 5. DoughanA.K & Dikalov S.I Antioxidants and Redox signaling 9. 1825-33 (2007) 6. Choi D-Y et al. Nature precedings posted 12 June (2008)
B. MS103-112 for detection of specific subunits1. Keeney P. et al. J. Neuroscience 26. 5256-64 (2006) 2. Pagliani DJ et al. Cell 134. 112-123 (2008) 3. Martinvalet D. et al. Cell 133. 681-92 (2008) 4. Bernard G. et al. J. Cell Science120. 838-48 (207) 5. Rea LS. et al. PLOS 6 e78 (2008) 6. Stankov K. Thyroid 16 325-31 (2006)
C. MS130-133 Dipstick assays for Complex I amount and activity (released spring 2008)1. Pagliani DJ. et al. Cell 134. 112-123 (2008).
Complex I Products
Assays & KitsNEW MS141 Microplate Assay Kit for Complex I ActivityNEW MS142 Microplate Assay Kit for Complex I Quantity MS130 Dipstick Assay Kit for Complex I ActivityMS131 Dipstick Assay Kit for Human Complex I QuantityMS133 Dipstick Assay Kit for Rodent Complex I QuantityMS101 Complex I Immunocapture Kit
Monoclonal AntibodiesMS101c Complex I Immunocapture monoclonal antibodyMS103 Complex I subunit GRIM-19 monoclonal antibodyMS104 Complex I subunit NDUFS4 monoclonal antibodyMS105 Complex I subunit NDUFB8 monoclonal antibodyMS107 Complex I subunit NDUFB4 monoclonal antibodyMS108 Complex I subunit NDUFB6 monoclonal antibodyMS109 Complex I subunit 8 kDa monoclonal antibody kDaMS110 Complex I subunit NDUFS3 monoclonal antibodyMS111 Complex I subunit NDUFA9 monoclonal antibody MS112 Complex I subunit NDUFS3 monoclonal antibody |