Mitochondrial oxidative phosphorylation is the main system to efficiently supplying ATP for the eukaryotic cell. Electron transfer through the respiratory chain concomitantly translocates protons across the inner membrane at three coupling sites (complex I, III, and IV). Complex I (CI, NADH:ubiquinone oxidoreductase) provides ~40% of proton-motive force for the ATP synthesis, while CI is one of the major sites of reactive oxygen species (ROS) generation and is extremely vulnerable to oxidative stress. Thus CI dysfunction is implicated in a variety of mitochondrial diseases including heart failure, type 2 diabetes, and neurodegenerative diseases such as Parkinson's disease. Therefore, the elucidation of CI mechanisms is crucial for understanding these diseases and developing therapeutic strategies. However, CI has been one of the most challenging molecules to study its mechanisms and functions, because of the gigantic size (~900 kDa) and complexity of 45 different subunits and several cofactors including as many as 8 iron-sulfur clusters. The central fundamental question of how electron transfer is linked to vectorial proton translocation in CI still remains unanswered even 30 years since Peter Mitchell won the Nobel prize for his chemiosmotic theory. The long-term goal of this proposal is to elucidate the redox-coupled proton (H+) pump mechanism in CI. Previously, the PI made significant findings from a photoaffinity labeling study with fenpyroximate (a potent CI inhibitor that ND5, a transporter module membrane subunit, is involved in both ubiquinone(UQ)-binding and H+ translocation. This led us to start mutational study of the NuoL subunit (E. coli ND5 homolog). It has been predicted that in CI, redox chemistry drives proton translocation via an indirect (conformation-driven) coupling mechanism, but there was no testable details. We recently found the possibility that two major but tightly coupled functions, electron transfer (ET) and proton (H+) pump, could be decoupled by novel mutations in ND5. This strongly suggests that CI operates an indirect coupling mechanism. We hypothesize that NuoL(ND5) is the key player in the indirect conformation-driven coupling mechanism in CI.
Specific aims are:
Aim1. Analyze the relationship between ET and H+ pumping activities in a series of novel NuoL mutants.
Aim2. Elucidate conformational changes important for the indirect H+ pump coupling mechanism.
Aim3. Investigate the involvement of other transporter subunits NuoM(ND4) and NuoN(ND2) in indirect coupling. This project will provide a molecular level of understanding of indirect (conformation-driven) coupling process in CI.

Public Health Relevance

Mitochondrial complex I (CI) plays a central role in the cellular aerobic energy metabolism, and CI defects are implicated in a variety of human mitochondrial diseases including heart failure, type 2 diabetes, and neurodegenerative diseases such as Parkinson's disease. Yet, CI is one of the least well understood respiratory complexes due to its large size and complexity, and the basic knowledge of CI mechanisms is far behind and insufficient to understand CI diseases. Our research focus is to elucidate CI mechanisms in order to understand CI disease and potentially improve clinical diagnosis, and help to develop therapeutic strategies for CI dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097409-02
Application #
8334580
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2011-09-30
Project End
2016-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
2
Fiscal Year
2012
Total Cost
$300,015
Indirect Cost
$110,015
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Zhang, Yan; Yang, Chunyu; Dancis, Andrew et al. (2017) EPR studies of wild type and mutant Dre2 identify essential [2Fe--2S] and [4Fe--4S] clusters and their cysteine ligands. J Biochem 161:67-78
Narayanan, Madhavan; Sakyiama, Joseph A; Elguindy, Mahmoud M et al. (2016) Roles of subunit NuoL in the proton pumping coupling mechanism of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli. J Biochem 160:205-215
Tsang, Sabrina H; Wang, Ranran; Nakamaru-Ogiso, Eiko et al. (2016) The Oncogenic Small Tumor Antigen of Merkel Cell Polyomavirus Is an Iron-Sulfur Cluster Protein That Enhances Viral DNA Replication. J Virol 90:1544-56
Holt, Peter J; Efremov, Rouslan G; Nakamaru-Ogiso, Eiko et al. (2016) Reversible FMN dissociation from Escherichia coli respiratory complex I. Biochim Biophys Acta 1857:1777-1785
Elguindy, Mahmoud M; Nakamaru-Ogiso, Eiko (2015) Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2). J Biol Chem 290:20815-26
Narayanan, Madhavan; Leung, Steven A; Inaba, Yuta et al. (2015) Semiquinone intermediates are involved in the energy coupling mechanism of E. coli complex I. Biochim Biophys Acta 1847:681-9
McCormack, Shana; Polyak, Erzsebet; Ostrovsky, Julian et al. (2015) Pharmacologic targeting of sirtuin and PPAR signaling improves longevity and mitochondrial physiology in respiratory chain complex I mutant Caenorhabditis elegans. Mitochondrion 22:45-59
Frederick, David W; Davis, James G; Dávila Jr, Antonio et al. (2015) Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism. J Biol Chem 290:1546-58
Nakamaru-Ogiso, Eiko; Narayanan, Madhavan; Sakyiama, Joseph A (2014) Roles of semiquinone species in proton pumping mechanism by complex I. J Bioenerg Biomembr 46:269-77
Dingley, Stephen D; Polyak, Erzsebet; Ostrovsky, Julian et al. (2014) Mitochondrial DNA variant in COX1 subunit significantly alters energy metabolism of geographically divergent wild isolates in Caenorhabditis elegans. J Mol Biol 426:2199-216

Showing the most recent 10 out of 12 publications