Inverted Potentials Enhance Electron Bifurcation Efficiency Prior to Steady State

Electron bifurcation networks split electron pairs into strongly and weakly reducing pools at low thermodynamic cost. Bifurcating enzymes typically use two-electron cofactors with inverted reduction potentials. The advantages of inverted potentials remain unclear, as earlier studies on generic free energy landscapes showed that both normal and inverted potentials can support efficient steady-state bifurcation and confurcation. Here, we examine how potential inversion affects steady-state and pre-steady-state bifurcation and confurcation kinetics by modeling redox substrates as finite pools, to better represent finite biological systems. We confirm that both potential orderings support efficient steady-state bifurcation and confurcation. However, only inverted potentials suppress short-circuiting and reduce energy dissipation in the pre-steady-state regime of bifurcation and confurcation when the transport network is launched in an electron-depleted state. These findings suggest that when metabolism switches frequently between bifurcation and confurcation, where steady state is not maintained, inverted potentials at the bifurcating site confer an energetic advantage.