A simplifed functional version of the Escherichia coli sulfite reductase

Abstract

Escherichia coli sulfite reductase (SiR) is a large and soluble enzyme with an α8β4 quaternary structure. Protein α (or sulfite reductase flavoprotein) contains both FAD and FMN, whereas protein β (or sulfite reductase hemoprotein (SiR-HP)) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is set up to arrange the redox cofactors in a FAD-FMN-Fe4S4-Heme sequence to make an electron pathway between NADPH and sulfite. Whereas α spontaneously polymerizes, we have been able to produce SiR-FP60, a monomeric but fully active truncated version of it, lacking the N-terminal part (Zeghouf, M., Fontecave, M., Macherel, D., and Coves, J. (1998) Biochemistry 37, 6114-6123). Here we report the cloning, overproduction, and characterization of the β subunit. Pure recombinant SiR-HP behaves as a monomer in solution and is identical to the native protein in all its characteristics. Moreover, we demonstrate that the combination of SiR-FP60 and SiR-HP produces a functional 1:1 complex with tight interactions retaining about 20% of the activity of the native SiR. In addition, fully active SiR can be reconstituted by incubation of the octameric sulfite reductase flavoprotein with recombinant SiR-HP. Titration experiments and spectroscopic properties strongly suggest that the holoenzyme should be described as an α8β8 with equal amounts of α and β subunits and that the α8β4 structure is probobly not correct.