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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Qian
authors.forename: Catarina M
authors.initials: CM
authors.lastname: Paquete
authors.forename: Ricardo O
authors.initials: RO
authors.lastname: Louro
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Ross
authors.forename: Edward
authors.initials: E
authors.lastname: Labelle
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Bond
authors.forename: Ming
authors.initials: M
authors.lastname: Tien
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics; D004579:Electron Transport / Q000235:genetics; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry* / Q000235:genetics; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003580:Cytochromes; D005290:Ferric Compounds; D005296:Ferrous Compounds; D033862:Iron-Binding Proteins; D006418:Heme