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.
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.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.