John P. Hoben

Mechanistic insights into energy conservation by flavin-based electron bifurcation

The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. The unprecedented range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Understanding the broad substrate repertoire of nitroreductase based on its kinetic mechanism.

Background: Nitroreductase reduces a broad range of nitroaromatics. Results: Steady-state and pre-steady-state kinetics were combined with tests for aminoaromatic product formation. Conclusion: Both half-reactions occur via a simple mechanism lacking detectable gating steps consistent with the broad substrate repertoire. Significance: Nitroreductase does not generate p-aminobenzoic acid and, therefore, appears not to reduce nitro groups to amines. The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 °C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 ± 0.09 s−1, indicating one-step binding before the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s−1). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 700 ± 20 s−1 and a dissociation constant of 0.51 ± 0.04 mm. Thus, we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines.

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

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.