ATP Binding and a Second Reduction Enables a Conformationally Gated Uphill Electron Transfer

Uphill electron transfers allow reducing inert metabolites and activating low potential metalloenyzmes. One way to overcome the barrier of an uphill electron transfer is coupling to ATP hydrolysis. How the processes are coupled and how they produce a unidirectional transfer preventing the thermodynamically favorable back electron transfer is not understood. Here, we investigated the ATP-dependent electron transfer between the metallo-ATPase reductive activator of CoFeSP (RACo) and its B12-dependent partner protein CoFeSP. We show that electron transfer is triggered by ATP binding, overcoming a ΔE 0′ of more than −250 mV. Rapid electron transfer is conformationally gated and requires potassium or ammonium ions. Slow ATP hydrolysis delays the last reaction step to allow rereduction of the oxidized activator, shifting the electron-transfer equilibrium to the products. These insights into the mechanism provide us with a blueprint to efficiently harness the energy of ATP in a coupling scheme with conformational changes to generate a unidirectional uphill electron transfer.