Nucleotide Hydrolysis and Protein Conformational Changes in Azotobacter vinelandii Nitrogenase Iron Protein: Defining the Function of Aspartate 129
The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron tra...
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| Veröffentlicht in: | Biochemistry (Easton) 1995-08, Vol.34 (34), p.10713-10723 |
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| creator | Lanzilotta, William N Ryle, Matthew J Seefeldt, Lance C |
| description | The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 micromolar) was comparable to that found for wild-type Fe protein (128 micromolar). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 micromolar). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein ( -290 mV for wild-type Fe protein and-300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wildtype and D129E Fe proteins shifted |
| doi_str_mv | 10.1021/bi00034a003 |
| format | Article |
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The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 micromolar) was comparable to that found for wild-type Fe protein (128 micromolar). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 micromolar). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein ( -290 mV for wild-type Fe protein and-300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wildtype and D129E Fe proteins shifted</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi00034a003</identifier><identifier>PMID: 7662655</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>2,2'-Dipyridyl - metabolism ; 2,2'-Dipyridyl - pharmacology ; ACIDE ASPARTIQUE ; ACIDO ASPARTICO ; ADENOSINE TRIPHOSPHATE ; Adenosine Triphosphate - metabolism ; Adenosine Triphosphate - pharmacology ; ADENOSINTRIFOSFATO ; Aspartic Acid - metabolism ; AZOTE ; AZOTOBACTER VINELANDII ; Azotobacter vinelandii - enzymology ; Binding Sites ; Circular Dichroism ; Computer Graphics ; Electron Spin Resonance Spectroscopy ; Electron Transport ; Hydrolysis ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Mutagenesis, Site-Directed ; Nitrogen - metabolism ; NITROGENASA ; NITROGENASE ; Nitrogenase - chemistry ; Nitrogenase - genetics ; Nitrogenase - metabolism ; NITROGENO ; Oxidation-Reduction ; Oxidoreductases ; Protein Binding ; Protein Conformation ; REDUCCION ; REDUCTION</subject><ispartof>Biochemistry (Easton), 1995-08, Vol.34 (34), p.10713-10723</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a402t-7430ed7298f1626484c162589a2af24125cc0a8188a7eb0791abc6d0ac5f16583</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi00034a003$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi00034a003$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2764,27075,27923,27924,56737,56787</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/7662655$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lanzilotta, William N</creatorcontrib><creatorcontrib>Ryle, Matthew J</creatorcontrib><creatorcontrib>Seefeldt, Lance C</creatorcontrib><title>Nucleotide Hydrolysis and Protein Conformational Changes in Azotobacter vinelandii Nitrogenase Iron Protein: Defining the Function of Aspartate 129</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 micromolar) was comparable to that found for wild-type Fe protein (128 micromolar). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 micromolar). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein ( -290 mV for wild-type Fe protein and-300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wildtype and D129E Fe proteins shifted</description><subject>2,2'-Dipyridyl - metabolism</subject><subject>2,2'-Dipyridyl - pharmacology</subject><subject>ACIDE ASPARTIQUE</subject><subject>ACIDO ASPARTICO</subject><subject>ADENOSINE TRIPHOSPHATE</subject><subject>Adenosine Triphosphate - metabolism</subject><subject>Adenosine Triphosphate - pharmacology</subject><subject>ADENOSINTRIFOSFATO</subject><subject>Aspartic Acid - metabolism</subject><subject>AZOTE</subject><subject>AZOTOBACTER VINELANDII</subject><subject>Azotobacter vinelandii - enzymology</subject><subject>Binding Sites</subject><subject>Circular Dichroism</subject><subject>Computer Graphics</subject><subject>Electron Spin Resonance Spectroscopy</subject><subject>Electron Transport</subject><subject>Hydrolysis</subject><subject>Magnetic Resonance Spectroscopy</subject><subject>Molecular Structure</subject><subject>Mutagenesis, Site-Directed</subject><subject>Nitrogen - metabolism</subject><subject>NITROGENASA</subject><subject>NITROGENASE</subject><subject>Nitrogenase - chemistry</subject><subject>Nitrogenase - genetics</subject><subject>Nitrogenase - metabolism</subject><subject>NITROGENO</subject><subject>Oxidation-Reduction</subject><subject>Oxidoreductases</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>REDUCCION</subject><subject>REDUCTION</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkUFv1DAQhS0EKsvCiRsSkk9wQAE7G8cOt2WhtFK1VLSVerMmzmTrkrUX20Esf4M_jKssFQcuHtnvm6cZP0Kec_aWs5K_ay1jbFFBPh6QGRclK6qmEQ_JLL_XRdnU7DF5EuNtvlZMVkfkSNZ1WQsxI7_XoxnQJ9shPdl3wQ_7aCMF19Hz4BNaR1fe9T5sIVnvYKCrG3AbjDQry18--RZMwkB_WIdDbrOWrm0KfoMOItLT4N1fp_f0I_bWWbeh6Qbp8ejMnSf1PV3GHYQECSkvm6fkUQ9DxGeHOidXx58uVyfF2ZfPp6vlWQEVK1MhqwXDTpaN6nneplKVyVWoBkroy4qXwhgGiisFElsmGw6tqTsGRuQGoRZz8mry3QX_fcSY9NZGg0NeA_0YNZeMcSlEBt9MoAk-xoC93gW7hbDXnOm7CPQ_EWT65cF2bLfY3bOHP896Mek2Jvx5L0P4pmu5kEJfnl_o9QdVf2XXSl9n_sXE9-A1bIKN-uqikUypjM_J60kEE_WtH0OOKP53rD-wsaX2</recordid><startdate>19950829</startdate><enddate>19950829</enddate><creator>Lanzilotta, William N</creator><creator>Ryle, Matthew J</creator><creator>Seefeldt, Lance C</creator><general>American Chemical Society</general><scope>FBQ</scope><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>C1K</scope></search><sort><creationdate>19950829</creationdate><title>Nucleotide Hydrolysis and Protein Conformational Changes in Azotobacter vinelandii Nitrogenase Iron Protein: Defining the Function of Aspartate 129</title><author>Lanzilotta, William N ; Ryle, Matthew J ; Seefeldt, Lance C</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a402t-7430ed7298f1626484c162589a2af24125cc0a8188a7eb0791abc6d0ac5f16583</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><topic>2,2'-Dipyridyl - metabolism</topic><topic>2,2'-Dipyridyl - pharmacology</topic><topic>ACIDE ASPARTIQUE</topic><topic>ACIDO ASPARTICO</topic><topic>ADENOSINE TRIPHOSPHATE</topic><topic>Adenosine Triphosphate - metabolism</topic><topic>Adenosine Triphosphate - pharmacology</topic><topic>ADENOSINTRIFOSFATO</topic><topic>Aspartic Acid - metabolism</topic><topic>AZOTE</topic><topic>AZOTOBACTER VINELANDII</topic><topic>Azotobacter vinelandii - enzymology</topic><topic>Binding Sites</topic><topic>Circular Dichroism</topic><topic>Computer Graphics</topic><topic>Electron Spin Resonance Spectroscopy</topic><topic>Electron Transport</topic><topic>Hydrolysis</topic><topic>Magnetic Resonance Spectroscopy</topic><topic>Molecular Structure</topic><topic>Mutagenesis, Site-Directed</topic><topic>Nitrogen - metabolism</topic><topic>NITROGENASA</topic><topic>NITROGENASE</topic><topic>Nitrogenase - chemistry</topic><topic>Nitrogenase - genetics</topic><topic>Nitrogenase - metabolism</topic><topic>NITROGENO</topic><topic>Oxidation-Reduction</topic><topic>Oxidoreductases</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>REDUCCION</topic><topic>REDUCTION</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lanzilotta, William N</creatorcontrib><creatorcontrib>Ryle, Matthew J</creatorcontrib><creatorcontrib>Seefeldt, Lance C</creatorcontrib><collection>AGRIS</collection><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lanzilotta, William N</au><au>Ryle, Matthew J</au><au>Seefeldt, Lance C</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Nucleotide Hydrolysis and Protein Conformational Changes in Azotobacter vinelandii Nitrogenase Iron Protein: Defining the Function of Aspartate 129</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1995-08-29</date><risdate>1995</risdate><volume>34</volume><issue>34</issue><spage>10713</spage><epage>10723</epage><pages>10713-10723</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 micromolar) was comparable to that found for wild-type Fe protein (128 micromolar). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 micromolar). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein ( -290 mV for wild-type Fe protein and-300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wildtype and D129E Fe proteins shifted</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>7662655</pmid><doi>10.1021/bi00034a003</doi><tpages>11</tpages></addata></record> |
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| ispartof | Biochemistry (Easton), 1995-08, Vol.34 (34), p.10713-10723 |
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| source | MEDLINE; ACS Publications |
| subjects | 2,2'-Dipyridyl - metabolism 2,2'-Dipyridyl - pharmacology ACIDE ASPARTIQUE ACIDO ASPARTICO ADENOSINE TRIPHOSPHATE Adenosine Triphosphate - metabolism Adenosine Triphosphate - pharmacology ADENOSINTRIFOSFATO Aspartic Acid - metabolism AZOTE AZOTOBACTER VINELANDII Azotobacter vinelandii - enzymology Binding Sites Circular Dichroism Computer Graphics Electron Spin Resonance Spectroscopy Electron Transport Hydrolysis Magnetic Resonance Spectroscopy Molecular Structure Mutagenesis, Site-Directed Nitrogen - metabolism NITROGENASA NITROGENASE Nitrogenase - chemistry Nitrogenase - genetics Nitrogenase - metabolism NITROGENO Oxidation-Reduction Oxidoreductases Protein Binding Protein Conformation REDUCCION REDUCTION |
| title | Nucleotide Hydrolysis and Protein Conformational Changes in Azotobacter vinelandii Nitrogenase Iron Protein: Defining the Function of Aspartate 129 |
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