Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods

Bibliographic Details
Main Author: Franco, Ricardo
Publication Date: 1995
Other Authors: Moura, José J. G., Moura, Isabel, Lloyd, Steven G., Huynh, Boi Hanh, Forbes, William S., Ferreira, Glória C.
Format: Article
Language: eng
Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
Download full: https://doi.org/10.1074/jbc.270.44.26352
Summary: NIGMS NIH HHS (GM47295)
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spelling Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methodsBiochemistryMolecular BiologyCell BiologyNIGMS NIH HHS (GM47295)All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mössbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mössbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mössbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet. Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are δ = 1.36 ± 0.03 mm/s and ΔEQ = 3.04 ± 0.06 mm/s, distinct from the larger ΔEQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/ oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed.DQ - Departamento de QuímicaCQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE)RUNFranco, RicardoMoura, José J. G.Moura, IsabelLloyd, Steven G.Huynh, Boi HanhForbes, William S.Ferreira, Glória C.2019-09-10T22:49:24Z1995-11-031995-11-03T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article6application/pdfhttps://doi.org/10.1074/jbc.270.44.26352engFranco, R., Moura, J. J. G., Moura, I., Lloyd, S. G., Huynh, B. H., Forbes, W. S., & Ferreira, G. C. (1995). Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods. Journal of Biological Chemistry, 270(44), 26352-26357. https://doi.org/10.1074/jbc.270.44.263520021-9258PURE: 14632207http://www.scopus.com/inward/record.url?scp=0028840289&partnerID=8YFLogxKhttps://doi.org/10.1074/jbc.270.44.26352info:eu-repo/semantics/openAccessreponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiainstacron:RCAAP2025-05-26T01:39:28Zoai:run.unl.pt:10362/80765Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-28T17:12:07.627460Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiafalse
dc.title.none.fl_str_mv Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods
title Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods
spellingShingle Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods
Franco, Ricardo
Biochemistry
Molecular Biology
Cell Biology
title_short Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods
title_full Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods
title_fullStr Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods
title_full_unstemmed Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods
title_sort Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods
author Franco, Ricardo
author_facet Franco, Ricardo
Moura, José J. G.
Moura, Isabel
Lloyd, Steven G.
Huynh, Boi Hanh
Forbes, William S.
Ferreira, Glória C.
author_role author
author2 Moura, José J. G.
Moura, Isabel
Lloyd, Steven G.
Huynh, Boi Hanh
Forbes, William S.
Ferreira, Glória C.
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv DQ - Departamento de Química
CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE)
RUN
dc.contributor.author.fl_str_mv Franco, Ricardo
Moura, José J. G.
Moura, Isabel
Lloyd, Steven G.
Huynh, Boi Hanh
Forbes, William S.
Ferreira, Glória C.
dc.subject.por.fl_str_mv Biochemistry
Molecular Biology
Cell Biology
topic Biochemistry
Molecular Biology
Cell Biology
description NIGMS NIH HHS (GM47295)
publishDate 1995
dc.date.none.fl_str_mv 1995-11-03
1995-11-03T00:00:00Z
2019-09-10T22:49:24Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://doi.org/10.1074/jbc.270.44.26352
url https://doi.org/10.1074/jbc.270.44.26352
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Franco, R., Moura, J. J. G., Moura, I., Lloyd, S. G., Huynh, B. H., Forbes, W. S., & Ferreira, G. C. (1995). Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods. Journal of Biological Chemistry, 270(44), 26352-26357. https://doi.org/10.1074/jbc.270.44.26352
0021-9258
PURE: 14632207
http://www.scopus.com/inward/record.url?scp=0028840289&partnerID=8YFLogxK
https://doi.org/10.1074/jbc.270.44.26352
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 6
application/pdf
dc.source.none.fl_str_mv reponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron:RCAAP
instname_str FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron_str RCAAP
institution RCAAP
reponame_str Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
collection Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
repository.name.fl_str_mv Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
repository.mail.fl_str_mv info@rcaap.pt
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