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Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry

Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry
Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry
Biotin synthase from Escherichia coli was analyzed by nanoflow electrospray ionization mass spectrometry. From solution conditions in which the protein is in its native state, a distribution of monomeric, dimeric, and tetrameric species was observed. The distribution of these species was sensitive to changes in ionic strength: in the positive ion spectrum, biotin synthase at low ionic strength (pH 7.0-8.5) yielded less than 10% dimer. The masses of the monomeric species were consistent with the presence of a [2Fe-2S] cluster with a mass difference of 175.3 Da from the apomonomer with one disulfide bond. Despite the molecular mass of the noncovalent dimer (77 kDa), it was possible to observe a dimeric species containing one iron-sulfur cluster in both positive and negative ion spectra. Additionally, observation of a series of charge states assigned to the apodimer indicated that binding of the iron-sulfur cluster was not required to maintain the dimer. Binding of Cu2+ to biotin synthase was also observed; in the presence of excess chelating agent, free metals were removed and the iron-sulfur cluster remained intact. Evidence for the coordination of the iron-sulfur cluster in biotin synthase was obtained in a tandem mass spectrometry experiment. A single charge state containing the cluster at m/z 2416.9 was isolated, and collision-induced dissociation resulted in sequential loss of sulfur and retention of Fe3+.
clostridium-pasteurianum rubredoxin, in-vitro, protein, binding, 2fe-2s, identification, quantitation, (2+), purification, ferredoxin
0003-2700
4154-4161
Hernandez, H.
3620ed9b-445b-4397-bb56-a64b93e3f56e
Hewitson, K.S.
152c3269-8b89-4978-86cb-96c0e803a9f7
Roach, P.
ca94060c-4443-482b-af3e-979243488ba9
Shaw, N.M.
2031f90c-2f59-4f0f-8ae1-4d149b6b7fb2
Baldwin, J.E.
0d6004dd-a6c5-46d1-923c-fac8b0d39f9c
Robinson, C.V.
4957b09a-8a55-4a0f-84a4-a400385e1cb5
Hernandez, H.
3620ed9b-445b-4397-bb56-a64b93e3f56e
Hewitson, K.S.
152c3269-8b89-4978-86cb-96c0e803a9f7
Roach, P.
ca94060c-4443-482b-af3e-979243488ba9
Shaw, N.M.
2031f90c-2f59-4f0f-8ae1-4d149b6b7fb2
Baldwin, J.E.
0d6004dd-a6c5-46d1-923c-fac8b0d39f9c
Robinson, C.V.
4957b09a-8a55-4a0f-84a4-a400385e1cb5

Hernandez, H., Hewitson, K.S., Roach, P., Shaw, N.M., Baldwin, J.E. and Robinson, C.V. (2001) Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry. Analytical Chemistry, 73 (17), 4154-4161. (doi:10.1021/ac0102664).

Record type: Article

Abstract

Biotin synthase from Escherichia coli was analyzed by nanoflow electrospray ionization mass spectrometry. From solution conditions in which the protein is in its native state, a distribution of monomeric, dimeric, and tetrameric species was observed. The distribution of these species was sensitive to changes in ionic strength: in the positive ion spectrum, biotin synthase at low ionic strength (pH 7.0-8.5) yielded less than 10% dimer. The masses of the monomeric species were consistent with the presence of a [2Fe-2S] cluster with a mass difference of 175.3 Da from the apomonomer with one disulfide bond. Despite the molecular mass of the noncovalent dimer (77 kDa), it was possible to observe a dimeric species containing one iron-sulfur cluster in both positive and negative ion spectra. Additionally, observation of a series of charge states assigned to the apodimer indicated that binding of the iron-sulfur cluster was not required to maintain the dimer. Binding of Cu2+ to biotin synthase was also observed; in the presence of excess chelating agent, free metals were removed and the iron-sulfur cluster remained intact. Evidence for the coordination of the iron-sulfur cluster in biotin synthase was obtained in a tandem mass spectrometry experiment. A single charge state containing the cluster at m/z 2416.9 was isolated, and collision-induced dissociation resulted in sequential loss of sulfur and retention of Fe3+.

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More information

Published date: 1 September 2001
Keywords: clostridium-pasteurianum rubredoxin, in-vitro, protein, binding, 2fe-2s, identification, quantitation, (2+), purification, ferredoxin

Identifiers

Local EPrints ID: 19521
URI: http://eprints.soton.ac.uk/id/eprint/19521
ISSN: 0003-2700
PURE UUID: 5e7d3d50-0cbb-4459-802f-19265bc04752
ORCID for P. Roach: ORCID iD orcid.org/0000-0001-9880-2877

Catalogue record

Date deposited: 16 Feb 2006
Last modified: 20 Jul 2019 01:07

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