title: The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly.
abstract: Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.
authors.affiliation: From the Department of Biological Science and Institute of Molecular Biophysics and.
authors.forename: Isabel
authors.initials: I
authors.lastname: Askenasy
authors.affiliation: From the Department of Biological Science and Institute of Molecular Biophysics and.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Pennington
authors.affiliation: the Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306.
authors.forename: Yeqing
authors.initials: Y
authors.lastname: Tao
authors.affiliation: the Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, the National High Magnetic Field Laboratory, Tallahassee, Florida 32310, and.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
authors.affiliation: the National High Magnetic Field Laboratory, Tallahassee, Florida 32310, and.
authors.forename: Nicolas L
authors.initials: NL
authors.lastname: Young
authors.affiliation: the Center for Synchrotron Radiation Research and Instrumentation and Department of Biological and Chemical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616.
authors.forename: Weifeng
authors.initials: W
authors.lastname: Shang
authors.affiliation: From the Department of Biological Science and Institute of Molecular Biophysics and mestroupe@bio.fsu.edu.
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Stroupe
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D053838:Scattering, Small Angle; D050864:Sulfite Reductase (NADPH) / Q000737:chemistry*; D014961:X-Ray Diffraction
chemical_list: D029968:Escherichia coli Proteins; D050864:Sulfite Reductase (NADPH)
keywords: Fourier transform mass spectrometry; electron transfer; electrospray ionization; hydrogen-deuterium exchange; ion cyclotron resonance; iron-sulfur cluster biogenesis; iron-sulfur protein; metalloenzyme; small angle x-ray scattering (SAXS); sulfite reductase