Mechanistic insights into energy conservation by flavin-based electron bifurcation

Abstract

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.

Figure 1 |. Pf NfnI structure and orientation of cofactors.

Pf NfnI is a heterodimer of 31 kDa NfnI-S and 53 kDa NfnI-L. NfnI-S contains S-FAD and a [2Fe-2S] cluster with an unusual Asp ligand. NfnI-L contains one FAD (L-FAD), which is the site of electron bifurcation, and two [4Fe-4S] clusters. The L-FAD proximal [4Fe-4S] cluster coordination includes an unusual Glu ligand. S-FAD and L-FAD bind NADH and NADPH, respectively. Ferredoxin (Fd) is proposed to bind near the distal [4Fe-4S] cluster of NfnI-L. Edge-to-edge distances between cofactors are given in Ångström (Å). Pf NfnI shows sequence and structural similarity to the flavin-based electron bifurcating enzyme from Thermotoga maritima (NfnAB, PDB 4YRY). Fd_ox, oxidized Fd; Fd_red, reduced Fd.

Figure 2 |. Exergonic branch for electron transfer between NADP(H) and NAD(H) in Pf NfnI.

(a) Electron-transfer pathway between L-FAD and S-FAD. Em8 values are shown for [2Fe-2S] (ref. 16) and S-FAD (determined in this work). (b) A stable neutral semiquinone (NSQ) species of S-FAD is formed by reductive titration with NADPH under steady-state conditions and correlates with a decrease in OX. The increase in NSQ (A620 nm) as a function of NADPH concentration is shown in Supplementary Figure 3. (c) EPR spectra from reduction with NADH or NADPH. a.u., arbitrary units. n = 1 experiment for b and c.

Figure 3 |. Endergonic electron transfer between NADP(H) and Fd in Pf NfnI.

(a) Key residues that act to orient and control flavin reactivity at the bifurcating flavin site. (b) Square wave voltammograms from Pf NfnI adsorbed to an electrode (top) and a protein-free electrode (bottom). The FAD signal arises from both flavins (n = 3 experiments). SHE, standard hydrogen electrode. The pH dependency of the signals is shown in Supplementary Figure 6. (c) Reduction with sodium dithionite (pH 8.8 = −488 mV; pH 10 = −510 mV) or Ti-citrate (scaled by 0.5) was used to assign the two [4Fe-4S] cluster signals. More negative potentials led to relative increases in intensity of the uniquely coordinated proximal [4Fe-4S] cluster signal (g = 2.04, 1.96 and 1.93). (d) TAS difference spectra of Pf NfnI treated with NADPH at a time delay of 3 ps. The L-FAD ASQ absorption (366 nm) and OX bleach (455 nm) are shown. Spectral contributions from the pump at 400 nm have been removed for clarity. (e) TAS kinetic traces (data, points; fit, solid lines) of L-FAD ASQ decay (top) and OX bleach recovery (bottom) are correlated in time (i.e., ASQ absorption arises from OX bleach) with half-lives of 10 ps (ASQ = 10.2 ± 0.2 ps, OX = 10.0 ± 0.7 ps). n = 1 experiment for c; n = 2 experiments for d and e.

Figure 4 |. NADP(H) binding affects both short and long range interactions in Pf NfnI.

HDX-MS shows proton exchange in backbone residues after NADPH binding (red, more change; blue, less change). Increased exchange (red) with NADP+ occurred at L-FAD toward the protein surface and along the interface between NfnI-S and NfnI-L. (b) Unbound Pf NfnI showing NADP(H) binding cavity with L-FAD (cyan) and N5 interaction region. NADP(H) binding causes a loop rearrangement (blue) as visualized by superimposition of secondary structure of the NADP(H) bound Pf NfnI. (c) NADP(H) binding cavity with bound NADPH (blue sticks) along with HDX changes (red) and structural rearrangements (blue).