Spectroscopic and electrochemical investigation of multi-electron catalysis in sulfite and nitrite reductase enzymes

• Main Author: Judd, Evan Thomas
• Language: en_US
• Published: 2016
• Subjects: Biochemistry; Nitrite reductase; Protein film voltammetry; Sulfite reductase
• Online Access: https://hdl.handle.net/2144/16321

Multi-electron multi-proton reactions form the basis of nearly every chemical reaction involved in energy storage and manipulation. Despite their importance, the basic properties of these chemical transformations, such as the details of how electron transfer and proton-coupled redox events that must occur during these reactions are controlled, remain poorly understood. The sulfite and nitrite reductase family of enzymes are responsible for carrying out the six-electron reduction of sulfite to sulfide and nitrite to ammonia, respectively. These enzymes play fundamental roles in microbial metabolism and are either dissimilatory or assimilatory in nature. Multi-electron multi-proton reactions are investigated by the study of the catalytic mechanisms of two enzymes that are structurally different, but carry out similarly complex chemistry: the dimeric multi heme cytochrome c nitrite reductase from Shewanella oneidensis and the monomeric siroheme and [4Fe-4S] cluster containing sulfite reductase from Mycobacterium tuberculosis. Employing protein electrochemistry the properties of electron transfer steps and proton-coupled redox steps that occur throughout the catalytic cycle of cytochrome c nitrite reductase during its reduction of substrate revealed the strategies employed by this enzyme. The results presented indicate the reduction of substrate by the enzyme occurs in a series of one electron steps rather than coupled two-electron transfers. Mutational analysis of active site amino acids reveals their role in governing proton coupled redox events, which likely involves a hydrogen bonding network consisting of these residues and water molecules. Additionally, steady state kinetics assays coupled to site-directed mutagenesis of M. tuberculosis sulfite reductase identify a tyrosine residue adjacent to the active site which partially controls substrate preference, by influencing the electronic environment of the active site siroheme cofactor. Stopped-flow absorbance spectroscopy and rapid freeze quench electron paramagnetic resonance studies provide a first glimpse of a potential reaction intermediate during reduction of sulfite by sulfite reductase. Overall, our fundamental understanding of how sulfite and nitrite reductase enzymes catalyze complex multi-electron multi-proton reactions is advanced, and insight into the different approaches Nature employs to govern such powerful chemistry is revealed.