A simplifed functional version of the Escherichia coli sulfite reductase.
Escherichia coli sulfite reductase (SiR) is a large and soluble enzyme with an alpha(8)beta(4) quaternary structure. Protein alpha (or sulfite reductase flavoprotein) contains both FAD and FMN, whereas protein beta (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-Fe(4)S(4)-Heme sequence to make an electron pathway between NADPH and sulfite. Whereas alpha 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 Covès, J. (1998) Biochemistry 37, 6114-6123). Here we report the cloning, overproduction, and characterization of the beta 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 alpha(8)beta(8) with equal amounts of alpha and beta subunits and that the alpha(8)beta(4) structure is probably not correct.
Zeghouf
M
M
Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, CEA-Grenoble, France.
Fontecave
M
M
Coves
J
J
D001483:Base Sequence; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors