Modular organization and identification of a mononuclear iron-binding site within the NifU protein |
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| Authors: | J N Agar P Yuvaniyama R F Jack V L Cash A D Smith D R Dean M K Johnson |
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| Institution: | (1) Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA Tel.: +1-540-2315895 Fax: +1-540-2317126 e-mail: deandr@vt.edu, US;(2) Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA Tel.: +1-706-5429378 Fax: +1-706-5422353 e-mail: johnson@sunchem.chem.uga.edu, US |
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| Abstract: | The NifS and NifU nitrogen fixation-specific gene products are required for the full activation of both the Fe-protein and
MoFe-protein of nitrogenase from Azotobacter vinelandii. Because the two nitrogenase component proteins both require the assembly of Fe-S]-containing clusters for their activation,
it has been suggested that NifS and NifU could have complementary functions in the mobilization of sulfur and iron necessary
for nitrogenase-specific Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and
can be used to supply sulfide for the in vitro catalytic formation of Fe-S] clusters. The NifU protein was previously purified
and shown to be a homodimer with a 2Fe-2S] cluster in each subunit. In the present work, primary sequence comparisons, amino
acid substitution experiments, and optical and resonance Raman spectroscopic characterization of recombinantly produced NifU
and NifU fragments are used to show that NifU has a modular structure. One module is contained in approximately the N-terminal
third of NifU and is shown to provide a labile rubredoxin-like ferric-binding site. Cysteine residues Cys35, Cys62, and Cys106 are necessary for binding iron in the rubredoxin-like mode and visible extinction coefficients indicate that up to one ferric
ion can be bound per NifU monomer. The second module is contained in approximately the C-terminal half of NifU and provides
the 2Fe-2S] cluster-binding site. Cysteine residues Cys137, Cys139, Cys172, and Cys175 provide ligands to the 2Fe-2S] cluster. The cysteines involved in ligating the mononuclear Fe in the rubredoxin-like site
and those that provide the 2Fe-2S] cluster ligands are all required for the full physiological function of NifU. The only
two other cysteines contained within NifU, Cys272 and Cys275, are not necessary for iron binding at either site, nor are they required for the full physiological function of NifU. The
results provide the basis for a model where iron bound in labile rubredoxin-like sites within NifU is used for Fe-S] cluster
formation. The 2Fe-2S] clusters contained within NifU are proposed to have a redox function involving the release of Fe from
bacterioferritin and/or the release of Fe or an Fe-S] cluster precursor from the rubredoxin-like binding site.
Received: 27 October 1999 / Accepted: 30 November 1999 |
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| Keywords: | Iron-sulfur clusters assembly Iron metabolism NifU protein Resonance Raman Rubredoxin |
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