Flavin-based electron bifurcation allows enzymes to redistribute energy among electrons by coupling endergonic and exergonic electron transfer reactions. Diverse bifurcating enzymes employ a two-flavin electron transfer flavoprotein (ETF) that accepts hydride from NADH at a flavin (the so-called bifurcating FAD, Bf-FAD). The Bf-FAD passes one electron exergonically to a second flavin thereby assuming a reactive semiquinone state able to reduce ferredoxin or flavodoxin semiquinone. The flavin that accepts one electron and passes it on via exergonic electron transfer is known as the electron transfer FAD (ET-FAD) and is believed to correspond to the single FAD present in canonical ETFs, in domain II. The Bf-FAD is believed to be the one that is unique to bifurcating ETFs, bound between domains I and III. This very reasonable model has yet to be challenged experimentally. Herein we used site-directed mutagenesis to disrupt FAD binding to the presumed Bf site between domains I and III, in the Bf-ETF from Rhodopseudomonas palustris (RpaETF). The resulting protein contained only 0.80 ± 0.05 FAD, plus 1.21 ± 0.04 bound AMP as in canonical ETFs. The flavin was not subject to reduction by NADH, confirming absence of Bf-FAD. The retained FAD displayed visible circular dichroism (CD) similar to that of the ET-FAD of RpaETF. Likewise, the mutant underwent two sequential one-electron reductions forming and then consuming anionic semiquinone, reproducing the reactivity of the ET-FAD. These data confirm that the retained FAD in domain II corresponds the ET-FAD. Quantum chemical calculations of the absorbance and CD spectra of each of WT RpaETF's two flavins reproduced the observed differences between their CD and absorbance signatures. The calculations for the flavin bound in domain II agreed better with the spectra of the ET-flavin, and those calculated based on the flavin between domains I and III agreed better with spectra of the Bf-flavin. Thus calculations independently confirm the locations of each flavin. We conclude that the site in domain II harbours the ET-FAD whereas the mutated site between domains I and III is the Bf-FAD site, confirming the accepted model by two different tests.
|Number of pages||11|
|State||Published - 2019|
Bibliographical noteFunding Information:
This work was supported as part of the Biological and Electron Transfer and Catalysis (BETCy) EFRC, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Basic Energy Sciences under Award DE-SC0012518, the National Sciences Foundation, Chemistry of Life Processes CHE-1808433, and the German Research Foundation to the ‘Unifying Concepts in Catalysis” Cluster of Excellence UniCat-EXC314. Additional support was provided by the Research Challenge Trust fund of Kentucky (NMR) and the Technische Universität-Berlin (MAM, PH and AFM). AFM thanks the Odenwaldstraße Society for hospitality while executing calculations, Mssrs. S. Kraus and J. Krauss for excellent IT support and Prof. E. C. Glazer for access to a spectropolarimeter.
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ASJC Scopus subject areas
- Chemistry (all)