Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Your Mendeley pairing has expired. Please reconnect
ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

The Aging-Associated Enzyme CLK-1 Is a Member of the Carboxylate-Bridged Diiron Family of Proteins

View Author Information
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
*To whom correspondence should be addressed. Phone: (617) 253-1892. Fax: (617) 258-8150. E-mail: [email protected]
Cite this: Biochemistry 2010, 49, 45, 9679–9681
Publication Date (Web):October 5, 2010
Copyright © 2010 American Chemical Society

    Article Views





    Other access options
    Supporting Info (1)»


    Abstract Image

    The aging-associated enzyme CLK-1 is proposed to be a member of the carboxylate-bridged diiron family of proteins. To evaluate this hypothesis and characterize the protein, we expressed soluble mouse CLK-1 (MCLK1) in Escherichia coli as a heterologous host. Using Mössbauer and EPR spectroscopy, we established that MCLK1 indeed belongs to this protein family. Biochemical analyses of the in vitro activity of MCLK1 with quinone substrates revealed that NADH can serve directly as a reductant for catalytic activation of dioxygen and substrate oxidation by the enzyme, with no requirement for an additional reductase protein component. The direct reaction of NADH with a diiron-containing oxidase enzyme has not previously been encountered for any member of the protein superfamily.

    Read this article

    To access this article, please review the available access options below.

    Get instant access

    Purchase Access

    Read this article for 48 hours. Check out below using your ACS ID or as a guest.


    Access through Your Institution

    You may have access to this article through your institution.

    Your institution does not have access to this content. You can change your affiliated institution below.

    Supporting Information

    Jump To

    Experimental procedures and Figures S1−S6. This material is available free of charge via the Internet at

    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system:

    Cited By

    This article is cited by 33 publications.

    1. Weixue Wang, Roxana E. Iacob, Rebecca P. Luoh, John R. Engen, and Stephen J. Lippard . Electron Transfer Control in Soluble Methane Monooxygenase. Journal of the American Chemical Society 2014, 136 (27) , 9754-9762.
    2. Tsai-Te Lu, Seung Jae Lee, Ulf-Peter Apfel, and Stephen J. Lippard . Aging-Associated Enzyme Human Clock-1: Substrate-Mediated Reduction of the Diiron Center for 5-Demethoxyubiquinone Hydroxylation. Biochemistry 2013, 52 (13) , 2236-2244.
    3. Lucas J. Bailey, Justin F. Acheson, Jason G. McCoy, Nathaniel L. Elsen, George N. Phillips, Jr., and Brian G. Fox . Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase. Biochemistry 2012, 51 (6) , 1101-1113.
    4. Loi H. Do and Stephen J. Lippard . Toward Functional Carboxylate-Bridged Diiron Protein Mimics: Achieving Structural Stability and Conformational Flexibility Using a Macrocylic Ligand Framework. Journal of the American Chemical Society 2011, 133 (27) , 10568-10581.
    5. Woon Ju Song and Stephen J. Lippard . Mechanistic Studies of Reactions of Peroxodiiron(III) Intermediates in T201 Variants of Toluene/o-Xylene Monooxygenase Hydroxylase. Biochemistry 2011, 50 (23) , 5391-5399.
    6. Lucie Gonzalez, Samuel Chau‐Duy Tam Vo, Bruno Faivre, Fabien Pierrel, Marc Fontecave, Djemel Hamdane, Murielle Lombard. Activation of Coq6p, a FAD Monooxygenase Involved in Coenzyme Q Biosynthesis, by Adrenodoxin Reductase/Ferredoxin. ChemBioChem 2024, 25 (5)
    7. Sining Wang, Akash Jain, Noelle Alexa Novales, Audrey N. Nashner, Fiona Tran, Catherine F. Clarke. Predicting and Understanding the Pathology of Single Nucleotide Variants in Human COQ Genes. Antioxidants 2022, 11 (12) , 2308.
    8. Mateusz Manicki, Halil Aydin, Luciano A. Abriata, Katherine A. Overmyer, Rachel M. Guerra, Joshua J. Coon, Matteo Dal Peraro, Adam Frost, David J. Pagliarini. Structure and functionality of a multimeric human COQ7:COQ9 complex. Molecular Cell 2022, 82 (22) , 4307-4323.e10.
    9. Olivia M. Manley, Han N. Phan, Allison K. Stewart, Dontae A. Mosley, Shan Xue, Lide Cha, Hongxia Bai, Veda C. Lightfoot, Pierson A. Rucker, Leonard Collins, Taufika Islam Williams, Wei-Chen Chang, Yisong Guo, Thomas M. Makris. Self-sacrificial tyrosine cleavage by an Fe:Mn oxygenase for the biosynthesis of para -aminobenzoate in Chlamydia trachomatis. Proceedings of the National Academy of Sciences 2022, 119 (39)
    10. Scott Latimer, Shea A. Keene, Lauren R. Stutts, Antoine Berger, Ann C. Bernert, Eric Soubeyrand, Janet Wright, Catherine F. Clarke, Anna K. Block, Thomas A. Colquhoun, Christian Elowsky, Alan Christensen, Mark A. Wilson, Gilles J. Basset. A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in Arabidopsis. Journal of Biological Chemistry 2021, 297 (5) , 101283.
    11. Guangyu E. Chen, Nathan B. P. Adams, Philip J. Jackson, Mark J. Dickman, C. Neil Hunter. How the O2-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls. Nature Plants 2021, 7 (3) , 365-375.
    12. Lauren J. Rajakovich, Bo Zhang, Molly J. McBride, Amie K. Boal, Carsten Krebs, J. Martin Bollinger. Emerging Structural and Functional Diversity in Proteins With Dioxygen-Reactive Dinuclear Transition Metal Cofactors. 2020, 215-250.
    13. Keiko Tsuganezawa, Katsuhiko Sekimata, Yukari Nakagawa, Rei Utata, Kana Nakamura, Naoko Ogawa, Hiroo Koyama, Mikako Shirouzu, Takehiro Fukami, Kiyoshi Kita, Akiko Tanaka. Identification of small molecule inhibitors of human COQ7. Bioorganic & Medicinal Chemistry 2020, 28 (1) , 115182.
    14. Ludovic Pelosi, Chau-Duy-Tam Vo, Sophie Saphia Abby, Laurent Loiseau, Bérengère Rascalou, Mahmoud Hajj Chehade, Bruno Faivre, Mathieu Goussé, Clothilde Chenal, Nadia Touati, Laurent Binet, David Cornu, Cameron David Fyfe, Marc Fontecave, Frédéric Barras, Murielle Lombard, Fabien Pierrel, . Ubiquinone Biosynthesis over the Entire O 2 Range: Characterization of a Conserved O 2 -Independent Pathway. mBio 2019, 10 (4)
    15. Danielle C. Lohman, Deniz Aydin, Helaina C. Von Bank, Robert W. Smith, Vanessa Linke, Erin Weisenhorn, Molly T. McDevitt, Paul Hutchins, Emily M. Wilkerson, Benjamin Wancewicz, Jason Russell, Matthew S. Stefely, Emily T. Beebe, Adam Jochem, Joshua J. Coon, Craig A. Bingman, Matteo Dal Peraro, David J. Pagliarini. An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis. Molecular Cell 2019, 73 (4) , 763-774.e10.
    16. Robert Crichton. Iron. 2019, 363-404.
    17. Jonathan A. Stefely, David J. Pagliarini. Biochemistry of Mitochondrial Coenzyme Q Biosynthesis. Trends in Biochemical Sciences 2017, 42 (10) , 824-843.
    18. Adwitiya Kar, Haley Beam, Megan B. Borror, Michael Luckow, Xiaoli Gao, Shane L. Rea, . CLD1 Reverses the Ubiquinone Insufficiency of Mutant cat5/coq7 in a Saccharomyces cerevisiae Model System. PLOS ONE 2016, 11 (9) , e0162165.
    19. Ludovic Pelosi, Anne-Lise Ducluzeau, Laurent Loiseau, Frédéric Barras, Dominique Schneider, Ivan Junier, Fabien Pierrel, . Evolution of Ubiquinone Biosynthesis: Multiple Proteobacterial Enzymes with Various Regioselectivities To Catalyze Three Contiguous Aromatic Hydroxylation Reactions. mSystems 2016, 1 (4)
    20. . The Essential Role of Iron in Biology. 2016, 22-70.
    21. Christoph Freyer, Henrik Stranneheim, Karin Naess, Arnaud Mourier, Andrea Felser, Camilla Maffezzini, Nicole Lesko, Helene Bruhn, Martin Engvall, Rolf Wibom, Michela Barbaro, Yvonne Hinze, Måns Magnusson, Robin Andeer, Rolf H Zetterström, Ulrika von Döbeln, Anna Wredenberg, Anna Wedell. Rescue of primary ubiquinone deficiency due to a novel COQ7 defect using 2,4–dihydroxybensoic acid. Journal of Medical Genetics 2015, 52 (11) , 779-783.
    22. Mohammad Ozeir, Ludovic Pelosi, Alexandre Ismail, Caroline Mellot-Draznieks, Marc Fontecave, Fabien Pierrel. Coq6 Is Responsible for the C4-deamination Reaction in Coenzyme Q Biosynthesis in Saccharomyces cerevisiae. Journal of Biological Chemistry 2015, 290 (40) , 24140-24151.
    23. Cuiwen H. He, Dylan S. Black, Theresa P.T. Nguyen, Charles Wang, Chandra Srinivasan, Catherine F. Clarke. Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2015, 1851 (9) , 1227-1239.
    24. Ferman A. Chavez, Jia Li. Iron: Models of Proteins with Dinuclear Active Sites. 2015, 1-22.
    25. Justin F. Acheson, Lucas J. Bailey, Nathaniel L. Elsen, Brian G. Fox. Structural basis for biomolecular recognition in overlapping binding sites in a diiron enzyme system. Nature Communications 2014, 5 (1)
    26. Danielle C. Lohman, Farhad Forouhar, Emily T. Beebe, Matthew S. Stefely, Catherine E. Minogue, Arne Ulbrich, Jonathan A. Stefely, Shravan Sukumar, Marta Luna-Sánchez, Adam Jochem, Scott Lew, Jayaraman Seetharaman, Rong Xiao, Huang Wang, Michael S. Westphall, Russell L. Wrobel, John K. Everett, Julie C. Mitchell, Luis C. López, Joshua J. Coon, Liang Tong, David J. Pagliarini. Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis. Proceedings of the National Academy of Sciences 2014, 111 (44)
    27. Anthony L. Moore, Tomoo Shiba, Luke Young, Shigeharu Harada, Kiyoshi Kita, Kikukatsu Ito. Unraveling the Heater: New Insights into the Structure of the Alternative Oxidase. Annual Review of Plant Biology 2013, 64 (1) , 637-663.
    28. Ying Wang, Siegfried Hekimi. Molecular genetics of ubiquinone biosynthesis in animals. Critical Reviews in Biochemistry and Molecular Biology 2013, 48 (1) , 69-88.
    29. Fernando Gomez, Ryoichi Saiki, Randall Chin, Chandra Srinivasan, Catherine F. Clarke. Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation. Gene 2012, 506 (1) , 106-116.
    30. Loi H. Do, Stephen J. Lippard. Evolution of strategies to prepare synthetic mimics of carboxylate-bridged diiron protein active sites. Journal of Inorganic Biochemistry 2011, 105 (12) , 1774-1785.
    31. Mohammad Ozeir, Ulrich Mühlenhoff, Holger Webert, Roland Lill, Marc Fontecave, Fabien Pierrel. Coenzyme Q Biosynthesis: Coq6 Is Required for the C5-Hydroxylation Reaction and Substrate Analogs Rescue Coq6 Deficiency. Chemistry & Biology 2011, 18 (9) , 1134-1142.
    32. Carsten Krebs, J Martin Bollinger, Squire J Booker. Cyanobacterial alkane biosynthesis further expands the catalytic repertoire of the ferritin-like ‘di-iron-carboxylate’ proteins. Current Opinion in Chemical Biology 2011, 15 (2) , 291-303.
    33. Donald M. Kurtz, Emily Boice, Jonathan D. Caranto, Rosanne E. Frederick, Cesar A. Masitas, Kyle D. Miner. Iron: Non‐Heme Proteins with Diiron‐Carboxylate Active Sites. 2004, 1-18.