Relating protein structure to function: how protein dynamics maximizes energy gained by electron transfer in an anaerobic energy conservation mechanism

Abstract

Reduced ferredoxin (Fd) plays a critical role in anaerobic metabolism by acting as an alternative source of energy to adenosine triphosphate (ATP). The reduction potential of Fd is low (-450 mV) making it difficult to reduce individually. However, it has recently been discovered that a unique mechanism known as electron bifurcation allows anaerobic organisms to reduce Fd without suffering a loss of energy. Electron bifurcation was originally discovered in complex III of the electron transport chain, and increased the efficiency of the proton motive force without an overall change in the electron flow, minimizing energy loss. EB accomplishes this is by coupling a favorable (exergonic) and unfavorable (endergonic) reduction reaction. The exergonic reaction produces a singly reduced cofactor with a sufficiently negative reduction potential to allow the endergonic process to proceed. This allows anaerobic organisms to couple the formation of NADH, with the reduction of Fd. A detail of interest in the bifurcating mechanism is how these enzymes regulate the flow of electrons down the exergonic and endergonic branches to prevent multiple electrons from traveling down the exergonic branch. It is hypothesized that changes in the protein conformation alter the distance between cofactors altering the rate of electron transfer. To fully understand how changes in a protein's conformation regulates electron transfer in electron bifurcation we used a suite of in-solution techniques, such as H/D exchange and chemical cross-linking coupled to mass spectrometry to characterize the structure and dynamics of the model bifurcating enzyme, NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn), during the different steps of electron bifurcation. Additionally we also set out to use these techniques to characterize the structure and dynamics of the nitrogenase systems in order to obtain biophysical evidence of negative cooperativity in the various nitrogenase systems.