Jamariya Howard / Chemistry & Biochemistry / Faculty Mentor: Crystal Cooper
F420H2:NADP+ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP+ to NADPH, using reduced F420 cofactor as the hydride donor. NADPH and the F420 cofactor, are linked to several metabolic pathways including glycolysis and methanogenesis within methanogenic and sulfate-reducing archaea. Previous steady-state kinetic data conducted on wtFno displayed a Lineweaver-Burk plot, which was downward and concave in curvature, indicative of negative cooperativity. The pre steady-state kinetic studies displayed biphasic kinetics with an initial burst phase followed by a subsequent slow phase. The amplitude of the burst phase revealed 50% F420 cofactor reduction, indicating half-site reactivity. These studies suggest that Fno regulates NADPH production within the cell. The goal of this project is to determine which amino acids are involved in communication at the subunit interface. We have identified four amino acids which are Arg186, Thr192, Ser190, and His133. We have designed a library of Fno variants to test our hypothesis. The generated Fno variants (R186Q, R186K, R186I, T192V, T192A, H133A, H133N and S190A) were then characterized using fluorescence binding, steady-state and pre steady-state kinetic experiments. Our studies revealed that unlike wtFno, the following Fno variants, R186Q, R186K, R186I, T192A, and S190A displayed no cooperativity. This suggests that R186, T192 and S190 are involved in subunit communication. The results are reported here.
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