Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation

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dc.contributor.authorJimenez-Vicente, Emilioen
dc.contributor.authorYang, Zhi-Yongen
dc.contributor.authorRay, W. Keithen
dc.contributor.authorEchavarri-Erasun, Carlosen
dc.contributor.authorCash, Valerie L.en
dc.contributor.authorRubio, Luis M.en
dc.contributor.authorSeefeldt, Lance C.en
dc.contributor.authorDean, Dennis R.en
dc.contributor.departmentBiochemistryen
dc.date.accessioned2019-09-06T13:59:19Zen
dc.date.available2019-09-06T13:59:19Zen
dc.date.issued2018-06-22en
dc.description.abstractNitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as assembly factors. Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein-assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein-NafY, MoFe protein-NifY, and MoFe protein-NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.en
dc.description.notesMass spectrometry resources used in this work are maintained in part through funding by the Fralin Life Science Institute, the Agricultural Experiment Station Hatch Program, and the McIntire-Stennis Program at Virginia Tech and are managed by Richard F. Helm.en
dc.description.sponsorshipFralin Life Science Institute; Agricultural Experiment Station Hatch Program; McIntire-Stennis Program at Virginia Techen
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1074/jbc.RA118.002994en
dc.identifier.eissn1083-351Xen
dc.identifier.issue25en
dc.identifier.pmid29724822en
dc.identifier.urihttp://hdl.handle.net/10919/93497en
dc.identifier.volume293en
dc.language.isoenen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectprotein assemblyen
dc.subjectnitrogenaseen
dc.subjectreductaseen
dc.subjectprotein purificationen
dc.subjectelectron paramagnetic resonance (EPR)en
dc.subjectMoFe proteinen
dc.subjectFeMo-cofactoren
dc.subjectP-clusteren
dc.subjectnitrogen fixationen
dc.titleSequential and differential interaction of assembly factors during nitrogenase MoFe protein maturationen
dc.title.serialJournal of Biological Chemistryen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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