Reaction Mechanism and Determinants for Efficient Catalysis by DszB, a Key Enzyme for Crude Oil Bio-desulfurization

Sulfur oxides emitted by the burning of fossil fuels are a major environmental hazard. Strict legislation limits the sulfur content in fuels to ultralow levels, only achieved through harsh chemical methods that produce massive amounts of CO 2 . Bio-desulfurization represents a more environmentally friendly alternative, with the bacteria Rhodococcus erythropolis IGTS8 eliminating sulfur from the most chemically recalcitrant organosulfur compounds through a set of reactions performed by four enzymes (DszA-D). Despite its potential, bio-desulfurization is still too slow for direct use in refineries. As such, there is an urgent need to develop faster enzymes for the 4S pathway. To help in this endeavor, we determine here the reaction mechanism of the rate-limiting enzyme DszB with quantum mechanics/molecular mechanics methods and clarify the fine molecular requisites for efficient catalysis. The first and rate-limiting step of the reaction is the protonation of 2'-hydroxybiphenyl-2-sulfinate by Cys27 with an activation barrier of 25.3 kcal.mol(-1) and follows an electrophilic aromatic substitution mechanism, releasing the SO2 group and forming the Cys27 thiolate and the hydroxybiphenyl product. Cys27 and SO2 then react with a water molecule, completing the reactional cycle, and release sulfur as HSO3-. The rate-limiting step of the reaction was replicated for 21 different conformations of DszB, enabling the clear identification and quantification of the most relevant active-site preorganization requisites for the reaction to occur through low barriers. Finally, we rationally identified the residues that can be mutated to design much more efficient, oil refinery-competent DszB variants.