From Negative to No Cooperativity: Effects of Mutations on Intersubunit Communication within F₄₂₀H₂:NADP⁺ Oxidoreductase Using Steady-State and Pre-Steady-State Kinetic Methods

F₄₂₀H₂:NADP⁺ oxidoreductase (Fno) catalyzes the reversible production of NADPH by transferring a hydride from the reduced F₄₂₀ cofactor to NADP⁺. Previous kinetic studies suggest that wild-type Fno (<i>wt</i>Fno) displays half-site reactivity and negative cooperativity, making Fno regulatory within methanogenic and sulfate-reducing archaea. These studies identified four amino acids; R186, T192, S190, and H133, as potential candidates involved in intersubunit communication due to their location either at or within close proximity to the interface of the dimer. Therefore, a library of Fno variants─R186K, R186Q, R186I, T192V, T192A, S190A, H133A, and H133N─was generated and characterized using binding, steady-state, and pre-steady-state kinetic experiments to understand their involvement in communication. The Hill coefficient for <i>wt</i>Fno was previously reported as 0.61 ± 0.03, while the R186K, R186Q, R186I, and T192V Fno variant values were close or equal to 1, indicating a switch to no cooperativity behavior. The S190A variant displayed increased Hill coefficients of 0.8 ± 0.1 when compared to <i>wt</i>Fno, showing that cooperativity was affected. The steady-state double reciprocal plots of the R186 variants, S190A, and T192V Fno variants were linear, which is indicative of no cooperativity, departing from the negative cooperativity shape displayed by <i>wt</i>Fno. Unlike <i>wt</i>Fno, the pre-steady-state kinetic experiments did not display half-site reactivity for the variants. Additionally, the hydride transfer step became rate-limiting in catalysis for the R186K Fno variant only. Our data suggest that negative cooperativity can be disrupted and that the amino acids R186, T192, and S190 are involved in intersubunit communication.