Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5′-phosphosulfate (APS) or 3′-phosphoadenosine 5′-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO3 −) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor.

APS reductase catalyzes the first committed step of reductive sulfate assimilation in pathogenic bacteria, including Mycobacterium tuberculosis, and is a promising target for drug development. We report the 2.7 A resolution crystal structure of Pseudomonas aeruginosa APS reductase in the thiosulfonate intermediate form of the catalytic cycle and with substrate bound. The structure, high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, and quantitative kinetic analysis, establish that the two chemically discrete steps of the overall reaction take place at distinct sites on the enzyme, mediated via conformational flexibility of the C-terminal 18 residues. The results address the mechanism by which sulfonucleotide reductases protect the covalent but labile enzyme-intermediate before release of sulfite by the protein cofactor thioredoxin. P. aeruginosa APS reductase contains an [4Fe-4S] cluster that is essential for catalysis. The structure reveals an unusual mode of cluster coordination by tandem cysteine residues and suggests how this arrangement might facilitate conformational change and cluster interaction with the substrate. Assimilatory 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductases are evolutionarily related, homologous enzymes that catalyze the same overall reaction, but do so in the absence of an [Fe-S] cluster. The APS reductase structure reveals adaptive use of a phosphate-binding loop for recognition of the APS O3' hydroxyl group, or the PAPS 3'-phosphate group.

Water is one of the most fundamental molecules in chemistry, biology and astrophysics. It exists as two distinct nuclear-spin isomers, para- and ortho-water, which do not interconvert in isolated molecules. The experimental challenges in preparing pure samples of the two isomers have thus far precluded a characterization of their individual chemical behavior. Capitalizing on recent advances in the electrostatic deflection of polar molecules, we separate the ground states of para- and ortho-water in a molecular beam to show that the two isomers exhibit different reactivities in a prototypical reaction with trapped diazenylium ions. Based on ab initio calculations and a modelling of the reaction kinetics using rotationally adiabatic capture theory, we rationalize this finding in terms of different rotational averaging of ion-dipole interactions during the reaction.