H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin.
Identifieur interne : 004882 ( Main/Exploration ); précédent : 004881; suivant : 004883H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin.
Auteurs : S. Dong [États-Unis] ; J A Ybe ; M H Hecht ; T G SpiroSource :
- Biochemistry [ 0006-2960 ] ; 1999.
Descripteurs français
- KwdFr :
- Arbres (génétique), Asparagine (composition chimique), Asparagine (génétique), Asparagine (métabolisme), Glutamine (composition chimique), Glutamine (génétique), Leucine (composition chimique), Leucine (génétique), Liaison hydrogène (MeSH), Mutagenèse dirigée (MeSH), Plastocyanine (composition chimique), Plastocyanine (génétique), Plastocyanine (métabolisme), Réaction de polymérisation en chaîne (MeSH), Sites de fixation (génétique), Spectrophotométrie UV (MeSH), Thréonine (composition chimique), Thréonine (génétique).
- MESH :
- composition chimique : Asparagine, Glutamine, Leucine, Plastocyanine, Thréonine.
- génétique : Arbres, Asparagine, Glutamine, Leucine, Plastocyanine, Sites de fixation, Thréonine.
- métabolisme : Asparagine, Plastocyanine.
- Liaison hydrogène, Mutagenèse dirigée, Réaction de polymérisation en chaîne, Spectrophotométrie UV.
English descriptors
- KwdEn :
- Asparagine (chemistry), Asparagine (genetics), Asparagine (metabolism), Binding Sites (genetics), Glutamine (chemistry), Glutamine (genetics), Hydrogen Bonding (MeSH), Leucine (chemistry), Leucine (genetics), Mutagenesis, Site-Directed (MeSH), Plastocyanin (chemistry), Plastocyanin (genetics), Plastocyanin (metabolism), Polymerase Chain Reaction (MeSH), Spectrophotometry, Ultraviolet (MeSH), Threonine (chemistry), Threonine (genetics), Trees (genetics).
- MESH :
- chemical , chemistry : Asparagine, Glutamine, Leucine, Plastocyanin, Threonine.
- chemical , genetics : Asparagine, Glutamine, Leucine, Plastocyanin, Threonine.
- chemical , metabolism : Asparagine, Plastocyanin.
- genetics : Binding Sites, Trees.
- Hydrogen Bonding, Mutagenesis, Site-Directed, Polymerase Chain Reaction, Spectrophotometry, Ultraviolet.
Abstract
Type I Cu proteins maintain a trigonal N2S coordination group (with weak axial ligation) in both oxidation states of the Cu2+/+ ion, thereby reducing the reorganization energy for electron transfer. Requirements for maintaining this coordination group were investigated in poplar plastocyanin (Pcy) by mutation of a conserved element of the type 1 architecture, an asparagine residue (Asn38) adjacent to one of the ligating histidines. The side chain of this asparagine forms an active site clasp via two H-bonds with the residue (Ser85) adjacent to the ligating cysteine (Cys84). In addition, the main chain NH of Asn38 donates an H-bond to the thiolate ligand. We have investigated the importance of these interactions by mutating Asn38 to Gln, Thr, and Leu. The mutant proteins are capable of folding and binding Cu2+, but the blue color fades; the rate of fading increases in the order Gln < Thr < Leu. The color is not restored by ferricyanide, showing that the protein is modified irreversibly, probably by oxidation of Cys84. The more stable mutants N38Q and N38T were characterized spectroscopically. The wild-type properties are slightly perturbed for N38Q, but N38T shows remarkable similarity to another type 1 Cu protein, azurin (Azu) from Pseudomonas aeruginosa. The Cu-S(Cys) bond is longer in Azu than in Pcy, and the NH H-bond to the ligating S atom is shorter. Molecular modeling suggests a similar effect for N38T because the threonine residue shifts toward Ser85 in order to avoid a steric clash and to optimize H-bonding. These results demonstrate that H-bonding adjacent to the type 1 site stabilizes an architecture which both modulates the electronic properties of the Cu, and suppresses side reactions of the cysteine ligand.
DOI: 10.1021/bi981999u
PubMed: 10079082
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
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Dong</name><affiliation wicri:level="4"><nlm:affiliation>Department of Chemistry, Princeton University, New Jersey 08544, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry, Princeton University, New Jersey 08544</wicri:regionArea><orgName type="university">Université de Princeton</orgName><placeName><settlement type="city">Princeton (New Jersey)</settlement><region type="state">New Jersey</region></placeName></affiliation></author><author><name sortKey="Ybe, J A" sort="Ybe, J A" uniqKey="Ybe J" first="J A" last="Ybe">J A Ybe</name></author><author><name sortKey="Hecht, M H" sort="Hecht, M H" uniqKey="Hecht M" first="M H" last="Hecht">M H Hecht</name></author><author><name sortKey="Spiro, T G" sort="Spiro, T G" uniqKey="Spiro T" first="T G" last="Spiro">T G Spiro</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1999">1999</date><idno type="RBID">pubmed:10079082</idno><idno type="pmid">10079082</idno><idno type="doi">10.1021/bi981999u</idno><idno type="wicri:Area/Main/Corpus">004930</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">004930</idno><idno type="wicri:Area/Main/Curation">004930</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">004930</idno><idno type="wicri:Area/Main/Exploration">004930</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin.</title><author><name sortKey="Dong, S" sort="Dong, S" uniqKey="Dong S" first="S" last="Dong">S. Dong</name><affiliation wicri:level="4"><nlm:affiliation>Department of Chemistry, Princeton University, New Jersey 08544, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry, Princeton University, New Jersey 08544</wicri:regionArea><orgName type="university">Université de Princeton</orgName><placeName><settlement type="city">Princeton (New Jersey)</settlement><region type="state">New Jersey</region></placeName></affiliation></author><author><name sortKey="Ybe, J A" sort="Ybe, J A" uniqKey="Ybe J" first="J A" last="Ybe">J A Ybe</name></author><author><name sortKey="Hecht, M H" sort="Hecht, M H" uniqKey="Hecht M" first="M H" last="Hecht">M H Hecht</name></author><author><name sortKey="Spiro, T G" sort="Spiro, T G" uniqKey="Spiro T" first="T G" last="Spiro">T G Spiro</name></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><imprint><date when="1999" type="published">1999</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Asparagine (chemistry)</term><term>Asparagine (genetics)</term><term>Asparagine (metabolism)</term><term>Binding Sites (genetics)</term><term>Glutamine (chemistry)</term><term>Glutamine (genetics)</term><term>Hydrogen Bonding (MeSH)</term><term>Leucine (chemistry)</term><term>Leucine (genetics)</term><term>Mutagenesis, Site-Directed (MeSH)</term><term>Plastocyanin (chemistry)</term><term>Plastocyanin (genetics)</term><term>Plastocyanin (metabolism)</term><term>Polymerase Chain Reaction (MeSH)</term><term>Spectrophotometry, Ultraviolet (MeSH)</term><term>Threonine (chemistry)</term><term>Threonine (genetics)</term><term>Trees (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Arbres (génétique)</term><term>Asparagine (composition chimique)</term><term>Asparagine (génétique)</term><term>Asparagine (métabolisme)</term><term>Glutamine (composition chimique)</term><term>Glutamine (génétique)</term><term>Leucine (composition chimique)</term><term>Leucine (génétique)</term><term>Liaison hydrogène (MeSH)</term><term>Mutagenèse dirigée (MeSH)</term><term>Plastocyanine (composition chimique)</term><term>Plastocyanine (génétique)</term><term>Plastocyanine (métabolisme)</term><term>Réaction de polymérisation en chaîne (MeSH)</term><term>Sites de fixation (génétique)</term><term>Spectrophotométrie UV (MeSH)</term><term>Thréonine (composition chimique)</term><term>Thréonine (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Asparagine</term><term>Glutamine</term><term>Leucine</term><term>Plastocyanin</term><term>Threonine</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Asparagine</term><term>Glutamine</term><term>Leucine</term><term>Plastocyanin</term><term>Threonine</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Asparagine</term><term>Plastocyanin</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Asparagine</term><term>Glutamine</term><term>Leucine</term><term>Plastocyanine</term><term>Thréonine</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Binding Sites</term><term>Trees</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Arbres</term><term>Asparagine</term><term>Glutamine</term><term>Leucine</term><term>Plastocyanine</term><term>Sites de fixation</term><term>Thréonine</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Asparagine</term><term>Plastocyanine</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Hydrogen Bonding</term><term>Mutagenesis, Site-Directed</term><term>Polymerase Chain Reaction</term><term>Spectrophotometry, Ultraviolet</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Liaison hydrogène</term><term>Mutagenèse dirigée</term><term>Réaction de polymérisation en chaîne</term><term>Spectrophotométrie UV</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Type I Cu proteins maintain a trigonal N2S coordination group (with weak axial ligation) in both oxidation states of the Cu2+/+ ion, thereby reducing the reorganization energy for electron transfer. Requirements for maintaining this coordination group were investigated in poplar plastocyanin (Pcy) by mutation of a conserved element of the type 1 architecture, an asparagine residue (Asn38) adjacent to one of the ligating histidines. The side chain of this asparagine forms an active site clasp via two H-bonds with the residue (Ser85) adjacent to the ligating cysteine (Cys84). In addition, the main chain NH of Asn38 donates an H-bond to the thiolate ligand. We have investigated the importance of these interactions by mutating Asn38 to Gln, Thr, and Leu. The mutant proteins are capable of folding and binding Cu2+, but the blue color fades; the rate of fading increases in the order Gln < Thr < Leu. The color is not restored by ferricyanide, showing that the protein is modified irreversibly, probably by oxidation of Cys84. The more stable mutants N38Q and N38T were characterized spectroscopically. The wild-type properties are slightly perturbed for N38Q, but N38T shows remarkable similarity to another type 1 Cu protein, azurin (Azu) from Pseudomonas aeruginosa. The Cu-S(Cys) bond is longer in Azu than in Pcy, and the NH H-bond to the ligating S atom is shorter. Molecular modeling suggests a similar effect for N38T because the threonine residue shifts toward Ser85 in order to avoid a steric clash and to optimize H-bonding. These results demonstrate that H-bonding adjacent to the type 1 site stabilizes an architecture which both modulates the electronic properties of the Cu, and suppresses side reactions of the cysteine ligand.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">10079082</PMID><DateCompleted><Year>1999</Year><Month>04</Month><Day>15</Day></DateCompleted><DateRevised><Year>2013</Year><Month>11</Month><Day>21</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0006-2960</ISSN><JournalIssue CitedMedium="Print"><Volume>38</Volume><Issue>11</Issue><PubDate><Year>1999</Year><Month>Mar</Month><Day>16</Day></PubDate></JournalIssue><Title>Biochemistry</Title><ISOAbbreviation>Biochemistry</ISOAbbreviation></Journal><ArticleTitle>H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin.</ArticleTitle><Pagination><MedlinePgn>3379-85</MedlinePgn></Pagination><Abstract><AbstractText>Type I Cu proteins maintain a trigonal N2S coordination group (with weak axial ligation) in both oxidation states of the Cu2+/+ ion, thereby reducing the reorganization energy for electron transfer. Requirements for maintaining this coordination group were investigated in poplar plastocyanin (Pcy) by mutation of a conserved element of the type 1 architecture, an asparagine residue (Asn38) adjacent to one of the ligating histidines. The side chain of this asparagine forms an active site clasp via two H-bonds with the residue (Ser85) adjacent to the ligating cysteine (Cys84). In addition, the main chain NH of Asn38 donates an H-bond to the thiolate ligand. We have investigated the importance of these interactions by mutating Asn38 to Gln, Thr, and Leu. The mutant proteins are capable of folding and binding Cu2+, but the blue color fades; the rate of fading increases in the order Gln < Thr < Leu. The color is not restored by ferricyanide, showing that the protein is modified irreversibly, probably by oxidation of Cys84. The more stable mutants N38Q and N38T were characterized spectroscopically. The wild-type properties are slightly perturbed for N38Q, but N38T shows remarkable similarity to another type 1 Cu protein, azurin (Azu) from Pseudomonas aeruginosa. The Cu-S(Cys) bond is longer in Azu than in Pcy, and the NH H-bond to the ligating S atom is shorter. Molecular modeling suggests a similar effect for N38T because the threonine residue shifts toward Ser85 in order to avoid a steric clash and to optimize H-bonding. These results demonstrate that H-bonding adjacent to the type 1 site stabilizes an architecture which both modulates the electronic properties of the Cu, and suppresses side reactions of the cysteine ligand.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Dong</LastName><ForeName>S</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Department of Chemistry, Princeton University, New Jersey 08544, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ybe</LastName><ForeName>J A</ForeName><Initials>JA</Initials></Author><Author ValidYN="Y"><LastName>Hecht</LastName><ForeName>M H</ForeName><Initials>MH</Initials></Author><Author ValidYN="Y"><LastName>Spiro</LastName><ForeName>T G</ForeName><Initials>TG</Initials></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>R01-GM13498</GrantID><Acronym>GM</Acronym><Agency>NIGMS NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>R29-GM47894</GrantID><Acronym>GM</Acronym><Agency>NIGMS NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013487">Research Support, U.S. Gov't, P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biochemistry</MedlineTA><NlmUniqueID>0370623</NlmUniqueID><ISSNLinking>0006-2960</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0RH81L854J</RegistryNumber><NameOfSubstance UI="D005973">Glutamine</NameOfSubstance></Chemical><Chemical><RegistryNumber>2ZD004190S</RegistryNumber><NameOfSubstance UI="D013912">Threonine</NameOfSubstance></Chemical><Chemical><RegistryNumber>7006-34-0</RegistryNumber><NameOfSubstance UI="D001216">Asparagine</NameOfSubstance></Chemical><Chemical><RegistryNumber>9014-09-9</RegistryNumber><NameOfSubstance UI="D010970">Plastocyanin</NameOfSubstance></Chemical><Chemical><RegistryNumber>GMW67QNF9C</RegistryNumber><NameOfSubstance UI="D007930">Leucine</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001216" MajorTopicYN="N">Asparagine</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D001665" MajorTopicYN="N">Binding Sites</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005973" MajorTopicYN="N">Glutamine</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006860" MajorTopicYN="N">Hydrogen Bonding</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007930" MajorTopicYN="N">Leucine</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D016297" MajorTopicYN="Y">Mutagenesis, Site-Directed</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010970" MajorTopicYN="N">Plastocyanin</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D016133" MajorTopicYN="N">Polymerase Chain Reaction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013056" MajorTopicYN="N">Spectrophotometry, Ultraviolet</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013912" MajorTopicYN="N">Threonine</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014197" MajorTopicYN="N">Trees</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>1999</Year><Month>3</Month><Day>17</Day></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>1999</Year><Month>3</Month><Day>17</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>1999</Year><Month>3</Month><Day>17</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">10079082</ArticleId><ArticleId IdType="doi">10.1021/bi981999u</ArticleId><ArticleId IdType="pii">bi981999u</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country><region><li>New Jersey</li></region><settlement><li>Princeton (New Jersey)</li></settlement><orgName><li>Université de Princeton</li></orgName></list><tree><noCountry><name sortKey="Hecht, M H" sort="Hecht, M H" uniqKey="Hecht M" first="M H" last="Hecht">M H Hecht</name><name sortKey="Spiro, T G" sort="Spiro, T G" uniqKey="Spiro T" first="T G" last="Spiro">T G Spiro</name><name sortKey="Ybe, J A" sort="Ybe, J A" uniqKey="Ybe J" first="J A" last="Ybe">J A Ybe</name></noCountry><country name="États-Unis"><region name="New Jersey"><name sortKey="Dong, S" sort="Dong, S" uniqKey="Dong S" first="S" last="Dong">S. 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