Sulfur dioxide production through fossil fuel combustion is a major contributor to atmospheric pollution. This molecule has been known to cause respiratory illness in humans and land deterioration through acid rain formation. Fortunately, the biodesulfurization pathway (BDS) in R. erythropolis and other bacterial species have shown promise in becoming a useful tool for the removal of sulfur from crude oil. However, improvements are still necessary for this pathway to become competitive and industrially applicable. Our current objectives are to rationally design the BDS pathway in R. erythropolis, starting with enzymes DszC and DszA. The project goal is to decrease substrate specificity in the pathway, allowing it to work on a larger selection of organosulfur compounds found in fossil fuels. Here we exhibit preliminary biochemical characterization of the second enzyme in the BDS pathway, DszA, a flavin dependent monooxygenase. Stopped‐flow spectrophotometric studies are being used to monitor the reduced flavin dissociation constant(Kd) as well as its rate of oxidation in the presence or absence of DszA and its native substrate, DBTO2. A 20.3 ± 0.451 μM Kd value was determined for the reduced form of flavin. As expected, flavin oxidation increases with oxygen concentration in the presence of DszA. The rate of flavin oxidation also increases in the presence of both DszA and DBTO2 compared to DszA alone, suggesting that the oxidation we observe is enzymatically mediated. Contrary to previous studies, which suggested the production of a flavin‐N5‐oxide intermediate during catalysis, we have not observed the formation of flavin‐N5‐oxide. This intermediate should be detectable through stopped‐flow experiments. Our work towards confirming our enzyme’s activity and ability to generate either the N5‐oxide or the better‐understood C4a‐(hydro)peroxyflavin intermediates is ongoing.Support or Funding InformationCalifornia State University Program for Education in BiotechnologyNIH Grant 5SC3GM122652