Dokument

Summary:
Flavin-based electron bifurcation (FBEB), discovered in 2008, is a novel mode of energy coupling in anaerobic bacteria and archaea. The complex of electron-transferring flavoprotein and butyryl-CoA dehydrogenase (Etf/Bcd) mediates the reduction of crotonyl-CoA (E0′=10 mV) by NADH (E0′= 320 mV) only in presence of ferredoxin (E0′= 420 mV). During electron bifurcation, the two electrons from NADH find their destination in two different directions; one goes exergonicllay to crotonyl-CoA and the other moves endergonically to ferredoxin. Repetition of this process yields butyryl-CoA and a second reduced ferredoxin. The latter reduces either protons to give hydrogen via a soluble hydrogenase or NAD+ via the membrane-bound ferredoxin-NAD+ reductase (Rnf). The thereby formed electrochemical Na+-gradient is used for ATP synthesis. In this thesis, I have studied the dissociable Etf/Bcd complex from Acidaminococcus fermentans. The crystal structure of the heterodimeric Etf revealed the presence two FAD molecules, each bound to one subunit. NAD+ binds near the FAD of the smaller β-subunit (-FAD). Upon stepwise addition of NADH to Etf, first the FAD of the α-subunit (α-FAD) was reduced to FADH via the stable anionic semiquinone (α-FAD•). The second equivalent NADH reduced -FAD.In the presence of Bcd, reduction to α-FAD• required a whole equivalent of NADH. During the bifurcation process, stepwise addition of Etf to Bcd increased the rate of NADH oxidation until a molar ratio of Etf:Bcd (tetramer) = 2 was reached. The non-dissociable clostridial Bcd/Etf complexes have the same composition. The optimal ratio of ferredoxin: Etf: tetrameric Bcd in the presence of hydrogenase was 4:2:1, suggesting that under steady state conditions ferredoxin shuttles between the semireduced (Fd─) and completely reduced states (Fd2─). Our postulated mechanism of electron bifurcation starts with the reduction of -FAD by NADH to FADH. Then α-FAD, which is located on a flexible domain, approaches and takes one electron to yield the stabilized semiquinone α-FAD•. The remaining highly reactive electron on -FADH• is not stabilized and immediately reduces ferredoxin. The α-FAD• transfers its electron further to Bcd. After repetition of the bifurcation, a second reduced ferredoxin is formed and Bcd gets a second electron to reduce crotonyl-CoA. FurtherI illustrate that the brownish ferredoxin can be replaced by the bright yellow flavodoxin in the bifurcation process. The colorless hydroquinone of flavodoxin (E0′= 420 mV) can be reoxidized by NAD+ via Rnf to its blue semiquinone form (E0′= 60 mV) and thus shuttles between the semiquinone and hydroquinone forms. I also investigated the very similar bifurcating Etf/Bcd complex from Megasphaera elsdenii. In older studies, an apparent reduction of crotonyl-CoA by NADH was achieved without the need of ferredoxin. I found that under aerobic conditions oxygen fulfilled the need of ferredoxin and was reduced to hydrogen peroxide (H2O2). The up to 50% inhibition of the rate of NADH oxidation by superoxide dismutase suggested that the slow reduction of oxygen to superoxide (O2•) was followed by a fast reduction of O2• to H2O2. Interestingly, the same rates of NADH oxidation were observed by replacing crotonyl-CoA with butyryl-CoA. We propose an oxidation of butyryl-CoA by oxygen followed by the bifurcating reduction of crotonyl-CoA by NADH. Hence, under air and in the presence of catalytic amounts of crotonyl-CoA or butyryl-CoA, Etf/Bcd acts as NADH oxidase

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