![Figure 6. Ferredoxin (Fd). (A) The sequence of the N-terminal Fd domain of the CarCDE and 393 the Ldh-EtfAB complexes. EtfA of the Ldh-EtfAB complex contains one [4Fe-4S] cluster 394 ligated with four cysteines marked in green. In comparison, the equivalent CarE of the 395 CarCDE complex possesses two [4Fe-4S] clusters marked in blue and red. The latter is shared 396 between CarCDE and Ldh-EtfAB. (B) The structure of the Ldh-EtfAB complex superimposed 397 with EtfA (green) including the Fd domain (dark green) calculated by Alphafold2. The Fd 398 domain interacts with EtfB via the segment linking the anchor of the EtfB arm (225-232) and 399 the terminal strand (203:207) of the central β-sheet and via the loop following strand 81:85. 400 The [4Fe-4S] cluster (in ball-and-stick) of the Fd domain was modelled based on the properly 401 positioned four cysteine sulfurs. (C) Accessibility of b-FAD. b-FAD is more shielded in Ldh-402 EtfAB (red) than in the superimposed CarCDE (blue) due to the reoriented EtfB protrusion 403 such that an arriving Fd might not find a docking site sufficiently close to b-FAD. The structure 404 of non-bifurcating EtfAB of C. propionicum (grey) (calculated by Alphafold2) was also 405 superimposed to visualize the position of the inserted segment (lightgreen). An interference 406 with an external Fd remains elusive. The approximate diameter of Fd is drawn as dashed circle. 407 408](https://www.researchgate.net/publication/358385632/figure/fig1/AS:11431281098745678@1669076189102/Ferredoxin-Fd-A-The-sequence-of-the-N-terminal-Fd-domain-of-the-CarCDE-and-393-the_Q320.jpg)
February 2022
·
38 Reads
Lactate oxidation with NAD ⁺ as electron acceptor is a highly endergonic reaction and some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron transferring proteins EtfA and EtfB. The electron cryo-microscopically (cryo-EM) characterized (Ldh-EtfAB) 2 complex of Acetobacterium woodii at 2.43 Å resolution consists of a mobile EtfAB shuttle located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle contact each other thereby forming the D (dehydrogenase conducting) state. The intermediary Asp37 and Asp139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. A plausible novel B (bifurcation conducting) state with the EtfAB base and shuttle FADs in a productive electron transfer distance was derived by integrating Alphafold2 calculations. Kinetic analysis of enzyme variants shed light on the connection between NAD binding/release and D-to-B state transition. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is based on the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was outlined.