Active/de‐active state transition of the mitochondrial complex I as revealed by specific sulfhydryl group labeling

Gavrikova, Eleonora V.; Vinogradov, Andrei D.

doi:10.1016/s0014-5793(99)00850-9

Cited by 72 publications

(67 citation statements)

References 19 publications

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“…Using IBTP labeling in conjunction with BN-PAGE enabled us to confirm the presence of free thiol residues on the matrix surface of respiratory Complexes I, II, and IV. Whereas the presence of reactive thiols on these complexes is known, their locations and roles are uncertain; IBTP labeling will help address these points (1,(42)(43)(44)(45). Using a combination of proteomics approaches, we have uncovered a number of candidate protein thiols that are particularly susceptible to thiol modification, and these are now being investigated as potential regulators or redox sensors in the response of mitochondria to oxidative stress.…”

Section: Discussionmentioning

confidence: 99%

Specific Modification of Mitochondrial Protein Thiols in Response to Oxidative Stress

Lin¹,

Hughes²,

Muratovska³

et al. 2002

Journal of Biological Chemistry

182

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Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.Changes in the thiol redox state of mitochondrial proteins are significant in a number of cellular processes including the permeability transition, cell death due to calcium loading and oxidative stress, the response of cells to nitric oxide, tumor necrosis factor signaling, commitment to apoptosis, and in regulating respiratory chain function (1-9). However the detailed mechanisms and the proteins involved are uncertain. This is partly because of the technical challenges presented by determining thiol modifications of proteins in general and the difficulties inherent in mitochondrial proteomics. Potential protein thiol alterations include formation of mixed disulfides or internal disulfides from vicinal dithiols, S-nitrosation, and the formation of higher oxidation states (10 -15). The differential reactivity of individual protein thiols and the range of lifetimes of altered redox states can act as signal sensors or transducers to influence mitochondrial function (13-17). Nitric oxide may be a particularly important regulator of mitochondrial protein thiols because it diffuses easily into mitochondria and partitions selectively into the lipid bilayer where it can modify otherwise inaccessible thiols (18 -20). Modification of protein thiols by nitric oxide most likely occurs...

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Section: Discussionmentioning

confidence: 99%

Specific Modification of Mitochondrial Protein Thiols in Response to Oxidative Stress

Lin¹,

Hughes²,

Muratovska³

et al. 2002

Journal of Biological Chemistry

182

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“…Subsequently, on the addition of NADH, they either reorganize around the bound cation, locking it into the site, or reform the higher energy catalytic states, returning to the catalytic cycle. Interestingly, a sulfhydryl group becomes accessible to derivatization by N-ethylmaleimide in deactive complex I and may form part of the nascent zinc binding site (68). Conversely, during turnover, the tightly controlled conformations of the catalytic intermediates are less susceptible to Zn 2ϩ binding, which is slowed considerably.…”

Section: Is Complex I Inhibition Relevant To Zn 2ϩmentioning

confidence: 99%

The Inhibition of Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase) by Zn2+

Sharpley

Hirst

2006

Journal of Biological Chemistry

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NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, membrane-bound enzyme. It is central to energy transduction, an important source of cellular reactive oxygen species, and its dysfunction is implicated in neurodegenerative and muscular diseases and in aging. Here, we describe the effects of Zn 2؉ on complex I to define whether complex I may contribute to mediating the pathological effects of zinc in states such as ischemia and to determine how Zn 2؉ can be used to probe the mechanism of complex I. Zn 2؉ inhibits complex I more strongly than Mg 2؉ , Ca 2؉ , Ba 2؉ , and Mn 2؉ to Cu 2؉ or Cd 2؉ . It does not inhibit NADH oxidation or intramolecular electron transfer, so it probably inhibits either proton transfer to bound quinone or proton translocation. Thus, zinc represents a new class of complex I inhibitor clearly distinct from the many ubiquinone site inhibitors. No evidence for increased superoxide production by zinc-inhibited complex I was detected. Zinc binding to complex I is mechanistically complicated. During catalysis, zinc binds slowly and progressively, but it binds rapidly and tightly to the resting state(s) of the enzyme. Reactivation of the inhibited enzyme upon the addition of EDTA is slow, and inhibition is only partially reversible. The IC 50 value for the Zn 2؉ inhibition of complex I is high (10 -50 M, depending on the enzyme state); therefore, complex I is unlikely to be a major site for zinc inhibition of the electron transport chain. However, the slow response of complex I to a change in Zn 2؉ concentration may enhance any physiological consequences.

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“…The dramatic difference between D-and A-form in their SH-group(s) reactivity has been employed to identify the peptide that is speci cally involved in A/D transition. After extensive alkylation of submitochondrial particles or puri ed Complex I by N-ethylmaleimide, the further speci c labeling of small peptide, most likely IP-15, is seen only in de-activated preparations (32).…”

Section: The Slow Active/de-active State Enzyme Transformation (A/d Tmentioning

confidence: 99%

The Mitochondrial Complex I: Progress in Understanding of Catalytic Properties

Vinogradov¹,

Grivennikova²

2001

IUBMB Life

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SummaryAlthough the mitochondrial Complex I has been extensively studied for over 4 decades, its catalytic properties in mitochondria are poorly understood. This review summarizes the data on standard and nonstandard kinetic parameters of the enzyme. The slow interconversion between active and deactivated forms of the mammalian Complex I (A/D transition) is described. We discuss the potential relevance of this transition for the regulation of NADH oxidation by the respiratory chain under physiological and pathophysiological conditions. IUBMB Life, 52: 129-134, 2001

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Active/de‐active state transition of the mitochondrial complex I as revealed by specific sulfhydryl group labeling

Cited by 72 publications

References 19 publications

Specific Modification of Mitochondrial Protein Thiols in Response to Oxidative Stress

Specific Modification of Mitochondrial Protein Thiols in Response to Oxidative Stress

The Inhibition of Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase) by Zn2+

The Mitochondrial Complex I: Progress in Understanding of Catalytic Properties

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