DOI: 10.1073/pnas.101119498. The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase

The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase

10.1073/pnas.101119498

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

It is known that an E146D site-directed variant of the Azotobacter vinelandii iron protein (Fe protein) is specifically defective in its ability to participate in iron-molybdenum cofactor (FeMoco) insertion. Molybdenum-iron protein (MoFe protein) from the strain expressing the E146D Fe protein is partially (≈45%) FeMoco deficient. The “free” FeMoco that is not inserted accumulates in the cell. We were able to insert this “free” FeMoco into the partially pure FeMoco-deficient MoFe protein. This insertion reaction required crude extract of the Δ nifHDK A. vinelandii strain CA12, Fe protein and MgATP. We used this as an assay to purify a required “insertion” protein. The purified protein was identified as GroEL, based on the molecular mass of its subunit (58.8 kDa), crossreaction with commercially available antibodies raised against E. coli GroEL, and its NH 2 -terminal polypeptide sequence. The NH 2 -terminal polypeptide sequence showed identity of up to 84% to GroEL from various organisms. Purified GroEL of A. vinelandii alone or in combination with MgATP and Fe protein did not support the FeMoco insertion into pure FeMoco-deficient MoFe protein, suggesting that there are still other proteins and/or factors missing. By using GroEL-containing extracts from a Δ nifHDK strain of A. vinelandii CA12 along with FeMoco, Fe protein, and MgATP, we were able to supply all required proteins and/or factors and obtained a fully active reconstituted E146D nifH MoFe protein. The involvement of the molecular chaperone GroEL in the insertion of a metal cluster into an apoprotein may have broad implications for the maturation of other metalloenzymes.

Bibliography

Ribbe, M. W., & Burgess, B. K. (2001). The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase. Proceedings of the National Academy of Sciences, 98(10), 5521â5525.

Authors 2

Markus W. Ribbe (first)
Barbara K. Burgess (additional)

References 54 Referenced 42

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Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:44 a.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 8:14 p.m.)
Indexed	4 weeks ago (Aug. 3, 2025, 7 p.m.)
Issued	24 years, 4 months ago (May 1, 2001)
Published	24 years, 4 months ago (May 1, 2001)
Published Online	24 years, 4 months ago (May 1, 2001)
Published Print	24 years, 3 months ago (May 8, 2001)

Funders 0

None

BibTeX

@article{Ribbe_2001, title={The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase}, volume={98}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.101119498}, DOI={10.1073/pnas.101119498}, number={10}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Ribbe, Markus W. and Burgess, Barbara K.}, year={2001}, month=may, pages={5521–5525} }

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      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}