Biochemistry 51, 9857–9868 (2012)

Credit: KYLE SMITH

Sulfite reductase (SiR) has a central role in sulfur geochemistry, reducing SO32− to S2−. The enzyme uses an Fe4S4 cluster and a siroheme cofactor to pass the necessary six electrons to the substrate, whereas some of the necessary six protons are thought to come from positively charged amino acids forming a 'cationic cage' around the substrate. To clarify how these components work together, Smith and Stroupe now report the enzymatic and structural characterization of five SiR mutants. As expected, alterations to residues in a loop that neighbors the active site and is known to undergo conformational changes upon substrate binding resulted in structural perturbations. Similarly, alterations to some of the residues postulated to be involved in the cationic cage impaired enzyme activity even though the protein structure was intact. Quantification of electrons inserted per sulfur product and analysis of hydroxylamine reduction, which requires only two electrons and two protons, then allowed the authors to identify the incoming substrate and two ordered water molecules as likely sources for the first three protons and to assign the specific order of the final three proton transfers, which are mediated by defined cationic cage residues. Analysis of the reaction products also confirmed that electrons can be transferred individually, in contrast to one proposed mechanism of transfer in pairs. These results provide a compelling deconstruction of SiR's complex reaction.