Crystal Structures and Kinetics of S-Nitrosoglutathione Reductase from Arabidopsis thaliana and Homo sapiens

Abstract

The number of proteins shown to be S-nitrosated in vivo is increasing steadily, highlighting the importance of this redox based post translational modification. S-nitrosoglutathione reductase (GSNOR) reduces S-nitrosoglutathione (GSNO) to GSNHOH, removing GSNO from the cytosol. GSNOR has been linked to asthma in humans and has roles in thermotolerance and disease resistance in the model organism Arabidopsis thaliana. I have studied structure by x-ray crystallography and kinetics by steady state measurements and isothermal titration calorimetry of recombinant GSNOR from both these organisms. I present several structures, including the novel structure of Arabidopsis thaliana GSNOR to 1.4 Å resolution and a ternary complex of human GSNOR with GSNO and NAD+ to 1.5 Å resolution. GSNOR’s apparent ability to reduce GSNO in an environment where the high ratio of NAD⁺/NADH should prevent reductive chemistry from occurring had been unexplained. I present steady state and isothermal titration calorimetry data that demonstrate that GSNOR preferentially binds NADH, with several fold higher affinity than NAD⁺, allowing the enzyme to selectively bind NADH and reduce GSNO.