The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes |
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Authors: | Wayne Vigil Jr. Jessica Tran Dimitri Niks Gerrit J. Schut Xiaoxuan Ge Michael W.W. Adams Russ Hille |
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Affiliation: | 1.Department of Biochemistry, University of California, Riverside, California, USA;2.Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA |
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Abstract: | The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅−, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH−, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized. |
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Keywords: | electron bifurcation electron-transferring flavoprotein rapid-reaction kinetics electron paramagnetic resonance |
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