A 3.3 Å‐Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability

Abstract

Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c1 was found to greatly enhance the interaction between cyt. b and cyt. c1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment.

Semirational engineering of an aldo–keto reductase KmAKR for overcoming trade‐offs between catalytic activity and thermostability

“…Overall, the T 50 15 and T m of the “best” mutant Km AKR M13 were 6.3 and 10.4°C higher than those of Km AKR M9 , respectively. The CD spectra analysis demonstrated that the “best” mutant Km AKR M13 retains more α ‐helixes as compared with Km AKR M9 at elevated temperature, implying that the increased stability of the α ‐helixes in Km AKR M13 is related with the significantly enhanced thermostability of the enzyme, which is consistent with the report that α ‐helix stabilization is an important feature of highly thermostable enzymes (Brito e Cunha et al, 2019; H. L. Liu & Wang, 2003; Zhu et al, 2020). Additionally, MD simulations of Km AKR M9 and Km AKR M13 performed at 60°C showed that Km AKR M13 retained its structural stability during the 20 ns simulations whereas Km AKR M9 began to lose its structural stability after 5 ns simulations (Figure 6).…”

The Unusual Homodimer of a Heme‐Copper Terminal Oxidase Allows Itself to Utilize Two Electron Donors

“…We originally hypothesized it would be caused by formation of a supercomplex between AaC c O and complex III, providing additional quinol binding sites for its direct oxidation bypassing cytochrome c . However, our structural study of the A. aeolicus complex III [20] does not support this hypothesis. Furthermore, our substantial image processing of the current purified sample does not suggest the existing of any potential supercomplex (Figure S2).…”

“…In our previous study of A. aeolicus complex III, we discovered an extra transmembrane helix of cyt. c 1 and several unique residues important for the thermostability of the complex [20] . Interestingly, we also found that subunit I of AaC c O possesses two additional C‐terminal transmembrane helices, THM13 and TMH14, in comparison with eukaryotic C c Os, or still one additional TMH when comparing with TtC c O, a thermophilic prokaryotic C c O.…”

“…We originally hypothesized it would be caused by formation of supercomplex between AaC c O and complex III, providing additional quinol binding sites to enable its direct oxidation bypass cytochrome c . However, by solving the structure of A. aeolicus complex III, [20] we did not find novel structural features to support this hypothesis. In addition, our previous study showed the ubiquinol oxidation activity of the potential supercomplex was insensitive to stigmatellin, the inhibitor of ubiquinol binding of complex III [19] .…”

The Unusual Homodimer of a Heme‐Copper Terminal Oxidase Allows Itself to Utilize Two Electron Donors

“…Based on cryo‐EM 3D classification using RELION [21] (Figure S2), we found two well‐aligned classes of particles existing in the current purified sample. The first class represents the structure of dimeric complex III reported by us before [20] and the second represents the structure of dimeric AaC c O. Our substantial image processing does not suggest the existing of any potential supercomplex in this sample.…”