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A new mechanistic model for an O 2-protected electron-bifurcating hydrogenase, Hnd from Desulfovibrio fructosovorans

Abstract : The genome of the sulfate-reducing and anaerobic bacterium Desulfovibrio fructosovorans encodes different hydrogenases. Among them is Hnd, a tetrameric cytoplasmic [FeFe] hydrogenase that has previously been described as an NADP-specific enzyme (Malki et al., 1995). In this study, we purified and characterized a recombinant Strep-tagged form of Hnd and demonstrated that it is an electron-bifurcating enzyme. Flavin-based electron-bifurcation is a mechanism that couples an exergonic redox reaction to an endergonic one allowing energy conservation in anaerobic microorganisms. One of the three ferredoxins of the bacterium, that was named FdxB, was also purified and characterized. It contains a low-potential (E m =-450 mV) [4Fe4S] cluster. We found that Hnd was not able to reduce NADP + , and that it catalyzes the simultaneous reduction of FdxB and NAD +. Moreover, Hnd is the first electron-bifurcating hydrogenase that retains activity when purified aerobically due to formation of an inactive state of its catalytic site protecting against O 2 damage (H inact). Hnd is highly active with the artificial redox partner (methyl viologen) and can perform the electron-bifurcation reaction to oxidize H 2 with a specific activity of 10 µmol of NADH/min/mg of enzyme. Surprisingly, the ratio between NADH and reduced FdxB varies over the reaction 2 with a decreasing amount of FdxB reduced per NADH produced, indicating a more complex mechanism than previously described. We proposed a new mechanistic model in which the ferredoxin is recycled at the hydrogenase catalytic subunit.
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https://hal-amu.archives-ouvertes.fr/hal-01928576
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Submitted on : Tuesday, November 20, 2018 - 4:00:31 PM
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Arlette Kpebe, Martino Benvenuti, Chloé Guendon, Amani Rebai, Victoria Fernandez, et al.. A new mechanistic model for an O 2-protected electron-bifurcating hydrogenase, Hnd from Desulfovibrio fructosovorans. Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier, 2018, 1859 (12), pp.1302 - 1312. ⟨10.1016/j.bbabio.2018.09.364⟩. ⟨hal-01928576⟩

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