Abstract: Electron bifurcation allows the coupling of thermodynamically uphill and downhill electron transfer reactions, using two-electron donor cofactors that have very different potentials for the removal of the first and second electron. Bifurcating electron transfer enzymes typically send one electron uphill and one electron downhill by similar energies, such that the overall reaction is spontaneous, but not wasteful with an overall modest free energy change. Recent experimental progress using the NADH-dependent reduced ferredoxin: NADP+ oxidoreductase I (NfnI) as a model bifurcating enzyme has allowed us to dissect the mechanism of flavin-based electron bifurcation in the framework of modern electron transfer theory. The first electron that exits the bifurcating flavin executes a positive free energy "uphill" reaction, and triggers a second thermodynamically spontaneous electron transfer reaction along a second pathway that moves electrons in the opposite direction and at a very different potential. The singly reduced products formed from the bifurcating flavin electron transfers are more than twenty angstroms away from each other. In Nfn, the second electron to leave the flavin is much more reducing than the first: the potentials are said to be "crossed." The key thermodynamics and kinetics features of electron bifurcation in Nfn are (1) spatially separated transfer pathways that diverge from a two-electron donor, (2) one thermodynamically uphill and one downhill redox pathway, with a large negative shift in the donor's reduction potential after departure of the first electron, and (3) electron tunneling and activation factors that enable bifurcation.
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