We used site directed mutagenesis to make C42A and C47A mutations. While the exact mechanism of metal(loid) reduction by GRLMR is currently unknown, we hypothesized that the C42A and C47A mutations would abolish GRLMR catalytic activity, with minimal perturbations to expression strength, intracellular solubility, or other factors that might confound experimental / control paired experiments. Figure shows a Coomassie stained Native-PAGE gel of purified the GRLMR-C42A-C47A (Ala-GRLMR) and GRLMR side-by-side. To assay activity of the C42A, C47A mutant, we used an in situ assay of SeO3 2-reduction capability. In this assay, a non-denaturing gel containing SeO3 2-reducing enzymes is stained with a combination of SeO3 2-and NADPH. The result of this assay is shown in Figure . The assay shows a band comprised of selenium precipitates in the GRLMR lane, whereas there is no evidence of selenium precipitates in for the Ala-GRLMR mutant. This comprises strong evidence that the mutant GRLMR is catalytically inactive.