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

• Published in: The Journal of biological chemistry Vol. 292; no. 34; pp. 14039 - 14049
• Main Authors: Hoben, John P., Lubner, Carolyn E., Ratzloff, Michael W., Schut, Gerrit J., Nguyen, Diep M.N., Hempel, Karl W., Adams, Michael W.W., King, Paul W., Miller, Anne-Frances
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.08.2017; American Society for Biochemistry and Molecular Biology
• Subjects: Apoenzymes - chemistry; Apoenzymes - genetics; Apoenzymes - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Benzoic Acid - chemistry; Benzoic Acid - metabolism; Biocatalysis; Bioenergetics; Desulfovibrio vulgaris - enzymology; electron bifurcation; electron transfer; Electron Transport; energetics; Enterobacter cloacae - enzymology; flavin; Flavin-Adenine Dinucleotide - analogs & derivatives; Flavin-Adenine Dinucleotide - chemistry; Flavin-Adenine Dinucleotide - metabolism; Flavodoxin - chemistry; Flavodoxin - genetics; Flavodoxin - metabolism; flavoprotein; fluorescence; Holoenzymes - chemistry; Holoenzymes - genetics; Holoenzymes - metabolism; Models, Molecular; Multienzyme Complexes - chemistry; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - genetics; NADH, NADPH Oxidoreductases - metabolism; Nitroreductases - chemistry; Nitroreductases - genetics; Nitroreductases - metabolism; ortho-Aminobenzoates - chemistry; ortho-Aminobenzoates - metabolism; Oxidation-Reduction; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Pyrococcus furiosus - enzymology; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Silent Mutation; solar (fuels), biofuels (including algae and biomass), bio-inspired, hydrogen and fuel cells; Thermus thermophilus - enzymology; transient absorption spectroscopy; electron bifurcation; electron transfer; fluorescence; flavoprotein; transient absorption spectroscopy; energetics; flavin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.794214

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.