The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD<sup/>

Abstract

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD⁺, we propose a catalytic cycle involving formation of an intermediary NAD⁺-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP⁺ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD⁺, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.

Keywords: EtfABCX; anaerobic physiology; archaea; bifurcation; bioenergetics; charge transfer; electron paramagnetic resonance (EPR); electron transport; extreme thermophile; flavin; flavoprotein; radical; semiquinone.

Figure 1.

Cofactor content and proposed pathway of electron flow in P. aerophilum EtfABCX and P. furiosus NfnI. Q, quinone; QH₂, reduced quinone; Fd_ox, oxidized ferredoxin; Fd_red, reduced ferredoxin.

Figure 2.

A, the multilayer circular plot presents interactions within the P. aerophilum EtfABCX complex captured by cross-linking (BS3). The most outer layer represents the protein sequence: EtfA is shown in gray, EtfB is purple, EtfC is green, and EtfX is pink. Yellow and purple histograms show the location of BS3 target residues (Lys and Ser) and the density of cross-links, respectively. Blue lines represent connections between subunits (intersubunit cross-links), and red lines highlight interactions within a single unit (intrasubunit cross-links). Cross-links maintaining less than 20 ppm error and a score higher than 3 (a score >2 is considered significant) are displayed. B, protein surface map of the complete P. aerophilum EtfABCX complex (for simplicity, only the AB dimer is presented) shows GEE (modification sites shown in red) and DnsCl (modification sites shown in blue) labels incorporated in to the native complex during short exposure to the two labeling reagents. Residues at the dimer interface highlighted in green correspond to labels integrated into the structure exclusively in the latest time points. Individual proteins are color-coded as follows: EtfB in orange and EtfA in tan. C, ribbon diagram of the MS-validated EtfABCX model. EtfA and EtfB are shown in light and dark blue, respectively, EtfC is teal, EtfX is magenta, the BF-FAD is green, the ET-FAD is yellow, and the QR-FAD is pink. Organic cofactors are shown using thick sticks to emphasize the redox-active portions and thin sticks to complete the structures. Iron–sulfur clusters are shown in orange (Fe) and yellow (S), and the menaquinone (purple) was modeled using the 2GMH (porcine electron transfer flavoprotein-ubiquinone oxidoreductase) structure with bound ubiquinone.

Figure 3.

A, slopes of Nernst plots confirm the 1 e⁻ nature of events in phases 1 and 2 and the 2 e⁻ nature of phase 3. Best linear fits are as follows: phase 1 (blue), 0.66x + 0.55 (R² = 0.997); phase 2 (black), 0.55x − 0.27 (R² = 0.999), and phase 3 (red), 0.91x + 0.05 (R² = 0.998). For the data in blue the “oxidized” flavin is OX, “reduced” flavin is the ASQ, and the dye is thionine. For the data in black the oxidized flavin is ASQ, reduced flavin is the HQ, and the dye is Nile blue. For the data in red, oxidized flavin is OX, reduced flavin is HQ, and the dye is safranin-O. B, proposed reduction events for each FAD cofactor in EtfAB and the measured potentials.

Figure 4.

Titration of P. aerophilum EtfAB (74 μm) (A) and EtfABCX (15 μm) (B) with NADH. Arrows (left to right) indicate formation and disappearance of the ASQ, disappearance of OX flavin absorbance, and the formation of a broad CT complex. C, anaerobic titration with NAD⁺ of P. aerophilum EtfAB (74 μm) reduced with excess titanium citrate. Arrows (left to right) indicate the disappearance of ASQ and the formation of a broad CT complex.

Figure 5.

EPR spectra (9.38 GHz CW) of P. aerophilum EtfAB and EtfABCX. The samples are as follows. AB AP, as-purified EtfAB; AB NADH, EtfAB treated with NADH; ABCX NADH, EtfABCX treated with NADH; AB DT, EtfAB treated with sodium dithionite; AB DT-NAD, EtfAB sequentially treated with sodium dithionite and NAD⁺; ABCX AP, EtfABCX as purified; ABCX NADH, EtfABCX treated with NADH. Top spectra (A–E) were recorded at 77 K, and bottom spectra (F and G) were recorded at 10 K.

Figure 6.

Proposed catalytic cycle of EtfABCX. The complex is depicted in its bifurcation-ready state in which the ET-FAD is in the ASQ state. In the first round of NADH oxidation, the BF-FAD is reduced to the HQ state and forms a CT complex with NAD⁺. The first electron is transferred to the ASQ-ET-FAD to generate the HQ state, leaving the ASQ-BF-FAD. The transfer of an electron to the low-potential branch [4Fe-4S] cluster triggers a protein conformational change (indicated by the asterisk) that then enables HQ-ET-FAD to reduce the FAD of EtfC with 1 e⁻ converting it to the NSQ state. The catalytic cycle continues with the oxidation of a second NADH and the transfer of electrons down the low- and high-potential branches leading to the reduction of Fd and MQ, respectively. Boxed with a blue dotted line are the complexes with NAD⁺ that could form CT complexes.