Abstract
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 of ASQ generated 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.
Keywords:
electron bifurcation; electron transfer; energetics; flavin; flavoprotein; fluorescence; transient absorption spectroscopy.
Publication types
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Comparative Study
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Apoenzymes / chemistry
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Apoenzymes / genetics
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Apoenzymes / metabolism
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism*
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Benzoic Acid / chemistry
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Benzoic Acid / metabolism
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Biocatalysis
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Desulfovibrio vulgaris / enzymology
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Electron Transport
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Enterobacter cloacae / enzymology
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Flavin-Adenine Dinucleotide / analogs & derivatives*
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Flavin-Adenine Dinucleotide / chemistry
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Flavin-Adenine Dinucleotide / metabolism
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Flavodoxin / chemistry
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Flavodoxin / genetics
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Flavodoxin / metabolism*
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Holoenzymes / chemistry
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Holoenzymes / genetics
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Holoenzymes / metabolism
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Models, Molecular*
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Multienzyme Complexes / chemistry
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Multienzyme Complexes / genetics
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Multienzyme Complexes / metabolism*
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NADH, NADPH Oxidoreductases / chemistry
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NADH, NADPH Oxidoreductases / genetics
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NADH, NADPH Oxidoreductases / metabolism*
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Nitroreductases / chemistry
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Nitroreductases / genetics
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Nitroreductases / metabolism*
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Oxidation-Reduction
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Oxidoreductases / chemistry
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Oxidoreductases / genetics
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Oxidoreductases / metabolism*
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Pyrococcus furiosus / enzymology
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Silent Mutation
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Thermus thermophilus / enzymology
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ortho-Aminobenzoates / chemistry
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ortho-Aminobenzoates / metabolism
Substances
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Apoenzymes
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Bacterial Proteins
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Flavodoxin
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Holoenzymes
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Multienzyme Complexes
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Recombinant Fusion Proteins
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Recombinant Proteins
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ortho-Aminobenzoates
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anthranilic acid
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Flavin-Adenine Dinucleotide
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flavin semiquinone
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Benzoic Acid
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fenamic acid
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Oxidoreductases
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ferredoxin-NAD+ reductase
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NADH oxidase
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NADH, NADPH Oxidoreductases
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Nitroreductases
Associated data
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PDB/1NOX.pdb
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PDB/1KQC.pdb
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PDB/5JFC.pdb
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PDB/1J8Q.pdb