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.

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

“…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.…”

“…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.…”

“…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] .…”

“…We previously reported that AaCcOcan use both reduced cytochrome c and quinol as electron donors. [19] We originally hypothesized it would be caused by formation of as upercomplex between AaCcOa nd complex III, providing additional quinol binding sites for its direct oxidation bypassing cytochrome c. However,o ur 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).…”

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

“…TheA aCcOs ample was enriched by anion exchange chromatography and further purified by size-exclusion chromatography,a nd the fractions showing ad ominant homogenous band around 242 kDa in Blue Native PAGE were used for subsequent cryo-EM experiments (Figure S1). 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.T he first class represents the structure of dimeric complex III reported by us before [20] and the second represents the structure of dimeric AaCcO. Our substantial image processing does not suggest the existing of any potential supercomplex in this sample.A fter in silico purification, the dimeric AaCcOs tructure was determined at af inal resolution of 3. a 3 and from heme a 3 to Cu B are 5.0 and 5.1 ,respectively.…”