Abstract
Sulfur reduction is a fundamental biological process in most bacteria and plants. As a model system we study the Escherichia coli assimilatory sulfite reductase (aSiR), an ∼800kD complex that catalyzes the concerted six-electron reduction of sulfite (SO3−2) to biologically useful sulfide (S−2). The complex consists of alpha and beta subunits, the flavoprotein and hemoprotein respectively. The active site lies in the hemoprotein subunit (SiRHP) that contains a siroheme cofactor coupled to a 4Fe-4S cluster used to shuttle electrons to the bound substrate.To investigate the structural importance of siroheme in SiRHP, we removed the cofactor by both mutagenic and auxotrophic methods. Electrophoretic mobility shift assays, circular dichroism, and dynamic light scattering were used to characterize changes in SiRHP lacking siroheme (apo SIRHP). In addition, the occupancy of the 4Fe-4S cluster was spectroscopically probed using an Ellman's reagent test. Apo SiRHP was found to exist as a tetramer instead of a monomer and no longer contained the 4Fe-4S cluster. Additionally, the apo SiRHP tetramer formed by the absence of siroheme was unable to bind the flavoprotein subunit required for functional aSiR assembly. These results suggest a possible mechanism for removing non-functional SiRHP subunits from the assembly pool within the cell.
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