Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1

Yufeng Qian, Catarina M. Paquete, Ricardo O. Louro, Daniel E. Ross, Edward Labelle, Daniel R. Bond, Ming Tien

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

In the model microbe Shewanella oneidensis, multi-heme proteins are utilized for respiratory metabolism where metals serve as the terminal electron acceptor. Among those is the periplasm-localized small tetraheme cytochrome (STC). STC has been extensively characterized structurally and electrochemically to which electron flow in and out of the protein has been modeled. However, until the present work, no kinetic studies have been performed to probe the route of electron flow or to determine the iron-binding site on STC. Using iron chelated by EDTA, NTA, or citrate, we have used chemical modification, site-directed mutagenesis along with isothermal titration calorimetry (ITC), and stopped-flow measurements to identify the iron binding site of STC. Chemical modifications of STC revealed that carboxyl groups on STC are involved in binding of EDTA-Fe 3+. Scanning mutagenesis was performed on Asp and Glu to probe the putative iron-binding site on STC. Two STC mutants (D21N; D80N) showed ∼70% decrease in observed electron transfer rate constant with EDTA-Fe 3+ from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe 3+ was further confirmed by a significant decrease (>10-fold) in iron binding affinity.

Original languageEnglish (US)
Pages (from-to)6217-6224
Number of pages8
JournalBiochemistry
Volume50
Issue number28
DOIs
StatePublished - Jul 19 2011

Fingerprint

Shewanella
Cytochromes
Iron
Binding Sites
Edetic Acid
Electrons
Mutagenesis
Chemical modification
Hemeproteins
Periplasm
Calorimetry
Kinetics
Flow measurement
Viperidae
Site-Directed Mutagenesis
Titration
Metabolism
Citric Acid
Rate constants
Metals

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Qian, Y., Paquete, C. M., Louro, R. O., Ross, D. E., Labelle, E., Bond, D. R., & Tien, M. (2011). Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1. Biochemistry, 50(28), 6217-6224. https://doi.org/10.1021/bi2005015
Qian, Yufeng ; Paquete, Catarina M. ; Louro, Ricardo O. ; Ross, Daniel E. ; Labelle, Edward ; Bond, Daniel R. ; Tien, Ming. / Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1. In: Biochemistry. 2011 ; Vol. 50, No. 28. pp. 6217-6224.
@article{be398b957f2b47aa95d963024e73f1bb,
title = "Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1",
abstract = "In the model microbe Shewanella oneidensis, multi-heme proteins are utilized for respiratory metabolism where metals serve as the terminal electron acceptor. Among those is the periplasm-localized small tetraheme cytochrome (STC). STC has been extensively characterized structurally and electrochemically to which electron flow in and out of the protein has been modeled. However, until the present work, no kinetic studies have been performed to probe the route of electron flow or to determine the iron-binding site on STC. Using iron chelated by EDTA, NTA, or citrate, we have used chemical modification, site-directed mutagenesis along with isothermal titration calorimetry (ITC), and stopped-flow measurements to identify the iron binding site of STC. Chemical modifications of STC revealed that carboxyl groups on STC are involved in binding of EDTA-Fe 3+. Scanning mutagenesis was performed on Asp and Glu to probe the putative iron-binding site on STC. Two STC mutants (D21N; D80N) showed ∼70{\%} decrease in observed electron transfer rate constant with EDTA-Fe 3+ from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe 3+ was further confirmed by a significant decrease (>10-fold) in iron binding affinity.",
author = "Yufeng Qian and Paquete, {Catarina M.} and Louro, {Ricardo O.} and Ross, {Daniel E.} and Edward Labelle and Bond, {Daniel R.} and Ming Tien",
year = "2011",
month = "7",
day = "19",
doi = "10.1021/bi2005015",
language = "English (US)",
volume = "50",
pages = "6217--6224",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "28",

}

Qian, Y, Paquete, CM, Louro, RO, Ross, DE, Labelle, E, Bond, DR & Tien, M 2011, 'Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1', Biochemistry, vol. 50, no. 28, pp. 6217-6224. https://doi.org/10.1021/bi2005015

Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1. / Qian, Yufeng; Paquete, Catarina M.; Louro, Ricardo O.; Ross, Daniel E.; Labelle, Edward; Bond, Daniel R.; Tien, Ming.

In: Biochemistry, Vol. 50, No. 28, 19.07.2011, p. 6217-6224.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1

AU - Qian, Yufeng

AU - Paquete, Catarina M.

AU - Louro, Ricardo O.

AU - Ross, Daniel E.

AU - Labelle, Edward

AU - Bond, Daniel R.

AU - Tien, Ming

PY - 2011/7/19

Y1 - 2011/7/19

N2 - In the model microbe Shewanella oneidensis, multi-heme proteins are utilized for respiratory metabolism where metals serve as the terminal electron acceptor. Among those is the periplasm-localized small tetraheme cytochrome (STC). STC has been extensively characterized structurally and electrochemically to which electron flow in and out of the protein has been modeled. However, until the present work, no kinetic studies have been performed to probe the route of electron flow or to determine the iron-binding site on STC. Using iron chelated by EDTA, NTA, or citrate, we have used chemical modification, site-directed mutagenesis along with isothermal titration calorimetry (ITC), and stopped-flow measurements to identify the iron binding site of STC. Chemical modifications of STC revealed that carboxyl groups on STC are involved in binding of EDTA-Fe 3+. Scanning mutagenesis was performed on Asp and Glu to probe the putative iron-binding site on STC. Two STC mutants (D21N; D80N) showed ∼70% decrease in observed electron transfer rate constant with EDTA-Fe 3+ from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe 3+ was further confirmed by a significant decrease (>10-fold) in iron binding affinity.

AB - In the model microbe Shewanella oneidensis, multi-heme proteins are utilized for respiratory metabolism where metals serve as the terminal electron acceptor. Among those is the periplasm-localized small tetraheme cytochrome (STC). STC has been extensively characterized structurally and electrochemically to which electron flow in and out of the protein has been modeled. However, until the present work, no kinetic studies have been performed to probe the route of electron flow or to determine the iron-binding site on STC. Using iron chelated by EDTA, NTA, or citrate, we have used chemical modification, site-directed mutagenesis along with isothermal titration calorimetry (ITC), and stopped-flow measurements to identify the iron binding site of STC. Chemical modifications of STC revealed that carboxyl groups on STC are involved in binding of EDTA-Fe 3+. Scanning mutagenesis was performed on Asp and Glu to probe the putative iron-binding site on STC. Two STC mutants (D21N; D80N) showed ∼70% decrease in observed electron transfer rate constant with EDTA-Fe 3+ from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe 3+ was further confirmed by a significant decrease (>10-fold) in iron binding affinity.

UR - http://www.scopus.com/inward/record.url?scp=79960217415&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79960217415&partnerID=8YFLogxK

U2 - 10.1021/bi2005015

DO - 10.1021/bi2005015

M3 - Article

C2 - 21682327

AN - SCOPUS:79960217415

VL - 50

SP - 6217

EP - 6224

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 28

ER -

Qian Y, Paquete CM, Louro RO, Ross DE, Labelle E, Bond DR et al. Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1. Biochemistry. 2011 Jul 19;50(28):6217-6224. https://doi.org/10.1021/bi2005015