Equilibrium & ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation

John P. Hoben, Carolyn E. Lubner, Michael W. Ratzloff, Gerrit J. Schut, Diep M.N. Nguyen, Karl W. Hempel, Michael W.W. Adams, X. Paul W. King, X. Anne Frances Miller

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to "bifurcation." It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay ofASQgenerated photochemically in bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I and can be an indication of capacity for electron bifurcation.

Original languageEnglish
Pages (from-to)14039-14049
Number of pages11
JournalJournal of Biological Chemistry
Issue number34
StatePublished - Aug 25 2017

Bibliographical note

Funding Information:
This work is supported as part of the Biological and Electron Transfer and Catalysis (BETCy) EFRC, an Energy Frontier Research Center funded by the United States Department of Energy, Office of Science (DE-SC0012518). This work was also supported by the U. S. Department of Energy under Contract DE-AC36-08-GO28308 with the National Renewable Energy Lab-oratory (to C. E. L., M. W. R., and P. W. K.). The authors declare that they have no conflicts of interest with the contents of this article.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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