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 bc 1 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. c 1 was found to greatly enhance the interaction between cyt. b and cyt. c 1, 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.


Introduction
Cellular respiration complexes convert redox energy into at ransmembrane electrochemical proton gradient, which is used to synthesize adenosine triphosphate (ATP) or to transport various substances.I nt his process,t he cytochrome bc 1 complex (also known as complex III) plays akey role by catalyzing the electron transfer from quinols to cytochrome c simultaneously transporting protons across the membrane according to the "Q-cycle" mechanism. [1] At present, various structures of cytochrome bc 1 complexes from vertebrates, [2] yeast, [3] and a-proteobacteria [4] are available.T hey have provided many details that have helped to understand the structural arrangement and the catalytic mechanism of these protein assemblies.T he subunit composition of the complex varies between species, [2d, 3a, 4d] but three conserved core subunits are always present, namely cytochrome b (cyt. b)with the cofactor heme b L and heme b H ,cytochrome c 1 (cyt. c 1 )w ith cofactor heme c 1 ,a nd aR ieske iron-sulfur protein (ISP) with ab inuclear iron sulfur cluster (2Fe-2S). [2a, 5] Each cytochrome bc 1 complex contains two quinol/quinone binding sites,the oxidation site Q o and the reduction site Q i ,which are the targets for natural and designed inhibitors. [1d, 6] All cytochrome bc 1 complexes are nearly symmetric dimers. Our knowledge of cytochrome bc 1 complex structures has been so far limited to those from mesophilic species,solittle information is available on its structures from thermophiles, which hinders our understanding of its unique thermal stability allowing maintenance of the electron transfer reaction under extreme conditions.
Aquifex aeolicus is ah yperthermophilic chemoautotrophic e-proteobacterium with adaptive growth temperatures in the range of 85-95 8 8C. [7] As one of the most hyperthermophilic bacteria known, A. aeolicus is thought to be one of the oldest bacterial species.I ti sd istributed in hydrothermal environments on land and in oceans throughout the world, including hot compost piles or deep gold mines. A. aeolicus is recognized as the representative organism not only of the Aquifex genus but also of the Aquificaceae family and the order Aquificales. [8] In order to live at extremely high temperature, the proteins,n ucleic acids,l ipids,a nd other biomolecules of the organism must be adapted. This feature makes of A. aeolicus an ideal organism to study the structure and function of thermophilic proteins.
A. aeolicus is ac hemolithotrophic hydrogen oxidizer,s o its respiratory chain complexes usually use hydrogen as the primary electron donor and oxygen as electron acceptor in order to provide energy for metabolism. In particular, its cytochrome bc 1 complex uses anaphthoquinone derivative,2-VI,VII-tetrahydromultiprenyl-1,4-naphthoquinone (NQ) (23), as special substrate for electron transfer,with NQ being reduced to NQH 2 by electrons from hydrogen oxidation in previous reactions. [9] Moreover,t his complex has as ignificantly increased stability at extremely high temperatures.I n the present study,u sing single-particle electron cryomicroscopy (cryo-EM), we determined the structure of the cytochrome bc 1 complex from A. aeolicus without and with an inhibitor at 3.3 resolution. These structures not only reveal the precise arrangement of the bc 1 core subunits but also provide information about the causes for its thermostability and suggest possible mechanisms by which electrons are transferred in extreme thermal environments.

Structure Determination and Overall Structure of Cytochrome bc 1 Complex
Thec ytochrome bc 1 complex from A. aeolicus was solubilized with dodecyl b-d-maltoside from membranes.T he sample contains am ixture of complex III and complex IV as identified by laser-induced liquid bead ion desorption (LIL-BID) MS (Supporting Information, Figure S1). [10] A. aeolicus complex III contains all three core catalytic subunits:c yt. b with heme b L and heme b H ,c yt. c 1 with heme c 1 and the ISP with the binuclear Fe-S cluster (2Fe-2S). [11] Thes tructure of Abstract: Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to al acko fa tomic structures of cytochrome bc 1 complex (Complex III) from thermophilic bacteria, little is knownabout the adaptations of this macromolecular machine to hyperthermophilic environments.I nt his study,w ep urified the cytochrome bc 1 complex of Aquifex aeolicus,o ne of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure.Anextra transmembrane helix at the N-terminus of cyt. c 1 was found to greatly enhance the interaction between cyt. band cyt. c 1 ,and to bind ap hospholipid molecule to stabilizet he complex in the membrane.T hese results providet he structural basis for the hyperstability of the cytochrome bc 1 complex in an extreme thermal environment.
complex III was determined after high-resolution refinement with 93 622 particles.T he overall structure of the dimeric cytochrome bc 1 complex reaches aresolution of 3.3 according to the gold standard FSC 0.143 (Fourier Shell Correlation) criterion (Figures S2 A-E and S3). It is the first structure of ar espiratory chain complex from A. aeolicus,a swell as the first of a1 ,4-naphthoquinol oxidizing cytochrome bc 1 complex. It has dimensions of ca. 87 in height and 80 in length ( Figure 1A). In this map,m odels for the three core subunits of cyt. b,c yt. c 1 and ISP,t ogether with their cofactors (hemes b H , b L , c 1 ,2 Fe-2S cluster) and substrates (a 1,4-naphthoquinone), could be built ( Figure 1B and Figure S4). All three subunits form a C 2 -symmetric dimeric structure through the interaction of ac yt. bdimer.
1,4-Naphthoquinones Are Involved in the "Q-Cycle" In the classic Q-cycle mechanism, ac omplete enzymatic reaction of the cytochrome bc 1 complex involves two cycles, in which two ubiquinols (UQH 2 )a re oxidized in the Q o site. Thef irst UQH 2 delivers one electron to cytochrome c 1 through the ISP,w hereas the second electron is transferred to aU Qi nt he Q i site leading to the formation of as emiquinone radical. Thetwo protons from UQH 2 are released to the external space.This process has to be repeated in order to generate as table doubly reduced and protonated UQ thus yielding aUQH 2 in the Q i site,which is released immediately and can be used as as ubstrate in the Q o site. [12] The cytochrome bc 1 complex from A. aeolicus has been shown to use a1 ,4-naphthoquinone (NQ), as as pecial substrate,t o mediate electron transfer. [9a] In our cryo-EM structure, several NQ molecules can be clearly traced in both the Q i site and on the cytoplasmic side,but none are around the Q o site.I np articular, aw ell-defined density map for aN Qi s clearly visible in the Q i pocket formed by TMH1, TMH4 and TMH5 of the cyt. b ( Figure 1C). This observation makes good sense because NQ is not as ubstrate,b ut ap roduct at the Q o site.I ts tight binding might lead to product inhibition. The edge-to-edge distance between heme b H and NQ in the Q i site is 6.1 ,this short distance ensures efficient electron transfer from heme b H to NQ.
We also determined the cryo-EM structure of the cytochrome bc 1 complex from A. aeolicus with the Q i site inhibitor antimycin A( AMY). Antimycin Aw as found to block the electron transfer from the high spin heme b H to the quinone or semiquinone.The entire structure of the inhibited enzyme complex is identical with the native structure,except for the Q i site ( Figure 1D). In the native state,a1,4naphthoquinone molecule is located near heme b H ,b ut in the structure with the inhibitor, it is replaced by AMY, suggesting that this inhibitor competitively occupies the Q i site and prevents the entry of the substrate NQ,thus blocking the electron transport and the entire respiratory chain reactions.
The Three Protein Subunits of the Cytochrome bc 1 Complex from A. aeolicus Thec yt. b subunit of A. aeolicus has 409 amino acid residues forming 13 helices,i ncluding eight transmembrane helices (TMHs), which interact in the membrane with two TMHs of cyt. c 1 and one TMH of the ISP ( Figure 1A). The two cyt. b protomers bind to each other mainly through TMH1 and TMH4 to form as table dimer ( Figure 2A). Its overall conformation is basically the same as that of other cyt. b subunits,w ith RMSD values around 2 ( Figure 2B). Thes ubunit cyt. c 1 of A. aeolicus contains 5h elices and one heme c 1 as cofactor, which is held by the conserved CXXCH motif with the residues Cys70, Cys73, and His74 ( Figure 2C). Several conserved hydrophobic residues,like Ile159, Met171, Leu136 and Phe158, also interact with heme c 1 .I mportantly, the N-terminal TMH1 of cyt. c 1 forms aunique structure that will be discussed later. Overall structure of the cytochrome bc 1 complex from Aquifex aeolicus. [17] A) The protein structure (ribbon model) of the cytochrome bc 1 complex is shown in cartoon representation in two different views. The subunits cyt. b,cyt. c 1 and ISP are colored in green, orange and purple, respectively. The scales are indicated besidest he model, and the grey square represents the cell membrane. B) Location of the cofactors and the substrates (1,4-naphthoquinone, NQ) of the cytochrome bc 1 complex is shown using cyan stick representation, while the proteins are transparent. The edge-to-edge and center-to-center distances between cofactors are provided. C) View into the Q i binding site with NQ in the native structure. D) View into the Q i binding site with antimycin A(AMY) in the inhibitory structure.
Thet hird subunit, the ISP,c ontains one TMH, and one functional domain containing the [2Fe-2S] cluster,a nd an interconnecting linker region. Around the 2Fe-2S cluster as eries of highly conserved coordinating residues can be identified including His138, His113, Cys111, Cys135, Cys116, and Cys137 which bind and stabilize the 2Fe-2S cluster ( Figure 2C). In our structure,t he 2Fe-2S cluster containing domain is found near cytochrome c 1 ,t he edge-to-edge distance between heme c 1 and the 2Fe-2S cluster is only 10 .T his result suggests that the electron transfer between these two cofactors of A. aeolicus can be rapid and efficient. Their relative positions and orientations are the same as in the corresponding subunits of Bos taurus ( Figure 2D).

The Sequence Characteristics of the Cytochrome bc 1 Complex of A. aeolicus
Theo verall structure of the cytochrome bc 1 complex of A. aeolicus is similar to other reported cytochrome bc 1 com-plexes,s uch as those from Rhodobacter sphaeroides (PDB entry:5 KLI) and B. taurus (PDB entry:1 BE3) ( Figure 3A). However,t he hyperthermophilic life of A. aeolicus may require adaptive changes of the amino acid sequences and structural characteristics of its important protein complexes. In order to study the adaptations of the cytochrome bc 1 complex to hyperthermophilic life,the amino acid sequences of the cytochrome bc 1 complex from different species,including thermophilic bacteria (A. aeolicus,H ydrogenivirga sp, Thermocrini salbus,Thermocrinis minervae,and Hydrogenobacter thermophilus), other prokaryotes (R. sphaeroides and Rhodobacter capsulatus)a nd eukaryotes (Saccharomyces cerevisiae,G allus gallus,O vis aries,B .taurus and Homo sapiens)w ere aligned and an evolutionary tree was constructed. Ther esults show that all three protein subunits of the cytochrome bc 1 complexes show obvious sequence differences between the three groups of species ( Figure 3A). The amino acid sequences of the cytochrome bc 1 complex protein subunits of all five thermophilic bacteria are closely related,

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Research Articles 346 www.angewandte.org suggesting that the same or similar changes of the amino acid sequences occurred during the adaptation to their thermophilic environment. Alternatively,the thermophilic cytochrome bc 1 complexes might have been acquired by horizontal gene transfer. Thehigh sequence similarity of the TMH1s of cyt. c 1 argues for this latter possibility.
According to the sequence alignment result, we have identified several unique residues that are present in the thermophilic bacteria but different in mesophilic species.I n the cyt. b subunit, the amino acid residues specific for the thermophilic species include Ty r38, Ty r61, Phe83, and Arg222 ( Figure 3B). In the cyt. c 1 subunit, the whole N-terminal TMH1 spanning 1-30 amino acids,i st otally missing in all mesophilic species ( Figure 3C). There are seven phenylalanine (Phe11-12, 19-21, 25, 29) and two tryptophan residues (Trp4, 24) located in this helix, forming au nique "WF-rich motif". Based on the cryo-EM structure of the cytochrome bc 1 complex from A. aeolicus,wewere able to analyze the important functions of these unique residues in thermophilic bacteria.
Heme b H and 1,4-Naphthoquinone Binding are Further Stabilized in A. aeolicus In the cyt. b subunit of the cytochrome bc 1 complex from A. aeolicus,heme b H interacts with residues of TMH1, TMH2, and TMH4. Theheme-Fecoordinating residues are identified as the highly conserved His105 and His217 for heme b H ( Figure 4A). These interactions and the spatial conformation of heme b H are the same in all the cyt. b proteins from different species,a nd serve to protect the electron transfer reaction at the Q i site ( Figure 4B,C). However,i nt he structure of the cytochrome bc 1 complex from A. aeolicus, the carboxyl groups of heme b H additionally interact with Ty r38 and Arg119 ( Figure 4A)l eading to as tabilization of heme b H binding.Itisworth noting that the carbonyl oxygen and phenyl-hydroxy group of Ty r38 bind to both carboxyl groups of heme b H .The tyrosine residue is highly conserved in the thermophilic bacteria ( Figure 3B)b ut replaced by at ryptophan residue in the mesophilic species.I nt he structure of the cytochrome bc 1 complex of R. sphaeroides (PDB entry 5KLI) or B. taurus (PDB entry 1BE3), Tr p45 or Tr p31 interact with only one of the two carboxyl groups of heme b H .T hus,t he additional interaction we observe probably stabilizes heme b H binding at high temperatures.
In our structure,t here are two 1,4-naphthoquinone molecules in each Q i site ( Figure 7A), one of them being located close to the heme b H molecule with an edge-to-edge distance of 6.1 ,allowing fast electron transfer. Theplane of the NQ head-group is almost perpendicular to the porphyrin plane of heme b H .Interestingly,the binding of this active NQ is stabilized by interactions with Glu254 of TMH5 and Arg222 from TMH4 of cyt. b. Importantly,Arg222 residue is replaced by ah istidine in R. sphaeroides and B. taurus ( Figure 3B). Compared with the short histidine residue,t he positively charged side chain of Arg222 is closer to the oxygen atom of NQ.Thus,inthe proteins from the hyperthermophilic species, Arg222 could stabilize the binding of the substrate NQ at the center Q i site,t oo rient NQ favourably and optimize the distance to heme b H .

More Stable TMHs and Increased Affinity with the Q-pool in A. aeolicus
In the dimeric structure of the cytochrome bc 1 complex of A. aeolicus,t wo cyt. b protomers bind to each other, mainly through their TMH regions.W efind that aresidue unique to the thermophiles,T yr61 of cyt. b,i si nvolved in this dimer interaction. Thep henyl-hydroxy of Ty r61 in TMH1 of one protomer binds to the nitrogen atom of Arg197 in TMH4 of

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Research Articles another protomer enhancing the interactions between the two cyt. b protomers ( Figure 5A). Moreover,T yr61 is also close to the carbonyl oxygen of Va l31 in TMH1 of the ISP subunit, which enhances the interactions between these two subunits in the complex. In the structure of cytochrome bc 1 complex from R. sphaeroides and B. taurus,t his tyrosine residue is replaced by ahistidine residue (His68 or His54), which cannot interact with the adjacent arginine residue in TMH4 of the other protomer ( Figure 5B,C). Ty r61 is highly conserved in all thermophilic bacteria ( Figure 3B). Consequently,T yr61 of cyt. b helps to stabilize the TMH region in the complex.
Interestingly,i no ur complex structure,t here is a1 ,4naphthoquinone molecule buried in ah ydrophobic pocket formed by TMH2 of cyt. b,T MH1 of the ISP and TMH2 of cyt. c 1 ( Figure 5A). This pocket is located away from both the Q i and Q o site but close to the phospholipid layer around the complex ( Figure 7B). This NQ molecule remained tightly bound to the cytochrome bc 1 complex of A. aeolicus throughout the protein purification process,and likely originates from the Q-pool in the phospholipid layer. This type of quinone binding has not been found in other cytochrome bc 1 structures ( Figure 5B,C). In the cytochrome bc 1 complex of A. aeolicus Glu204 and Lys207 of TMH2 of the cyt. c 1 subunit bind to one carbonyl oxygen of NQ,while Arg30 and Ty r26 from TMH1 of the ISP subunit bind to the other carbonyl oxygen of NQ. ThePhe83 on TMH2 of the cyt. b subunit and Met211 of the cyt. c 1 subunit interact with the benzene ring of NQ.A mong these residues,P he83 of cyt. b, ah ighly conserved residue in the thermophilic bacteria ( Figure 3B). In the complex structures of R. sphaeroides or B. taurus,t his phenylalanine residue is replaced by ag lycine or at yrosine residue, respectively,a nd the internal chemical environment of this pocket has also changed al ot. Therefore,n oq uinone molecules have been found inside these pockets in their structures.T his observation suggests that Phe83 of cyt. b enhances the binding of NQ to the cytochrome bc 1 complex.

TMH1 of cyt. c 1 Improves the Stability of the Complex in the Membrane
Thecyt. c 1 subunit of A. aeolicus possesses two TMHs,an N-terminal one and aC-terminal one.Onthe contrary,ina ll other known cytochrome bc 1 structures,t he cyt. c 1 subunit possesses only the C-terminally located TMH ( Figure 6A). Thesequence alignment shows that, this N-terminal TMH1 of the cyt. c 1 subunit of A. aeolicus contains ap henylalanine/ tryptophan rich (WF-rich) motif,w hich is found in all investigated thermophilic bacteria but is missing in the mesophilic species ( Figure 3C). In our structure,t his WFrich TMH1 binds to the hydrophobic surface of the TMH region of the complex in the phospholipid layer ( Figure 6B). Importantly,this kind of interaction leads to the formation of ad eep hydrophobic groove,w hich traps ap hospholipid molecule.T he hydrophilic head group binds to the positively charged region of the complex through its phosphate moiety, while its long hydrophobic tail is buried by as eries of hydrophobic residues on TMH1 of cyt. c 1 subunit, including Phe11, Leu17, Phe19, and Phe20. Other hydrophobic residues of TMH1 bind to the hydrophobic surface of cyt. b to stabilize this pocket, including Ty r4, Phe12, Leu23, Ty r24 and Phe25 ( Figure 6C). By calculating the surface area of the protein complex, we found that the addition of this TMH1 motif increases the interface between cyt. b and cyt. c 1 subunits by 60 %, from 1560 2 to 2494 2 .T herefore,t he unique Nterminal TMH1 of cyt. c 1 not only enhances the subunit interactions inside the cytochrome bc 1 complex but also sequesters ap hospholipid molecule inside the complex. This feature may lead to an additional stabilization of the complex in the membrane.

Discussion
Thes tructural and functional studies of respiratory complexes have continued for many years. [2e,3d, 13] However, there has been al ack of atomic structures of the complexes from thermophilic bacteria apart from the respiratory complex Iand the cytochrome ba 3 from Thermus thermophilus. [14]  In the present study,wepurified the cytochrome bc 1 complex from A. aeolicus and determined its structure at 3.3 resolution using single-particle cryo-EM. This structure reveals the conformations of the three core subunits,namely cyt. b,cyt. c 1 and the ISP,aswell as the mode of binding of the cofactors and of the 1,4-naphthoquinone substrate (Figure 1A). Ther elative locations and distances among hemes, the 2Fe-2S center and 1,4-naphthoquinones support the existence of aQcyclereaction mechanism in the respiratory chain of A. aeolicus ( Figure 1B). Using this structure,wehave identified several sequences and structural characteristics for thermophilic bacteria, which are not found in other structures and could protect the structure and catalytic activity of the complex at extremely high temperature.
On one hand, the cytochrome bc 1 complex from A. aeolicus has enhanced overall stability in the membrane.T yr61 of cyt. b interacts with both the ISP and another cyt. b protomer to form atighter complex ( Figure 5A). ANQsubstrate from the Q-pool is trapped inside ahydrophobic pocket and binds to as eries of hydrophobic and hydrophilic residues in the three subunits of the cytochrome bc 1 complex ( Figure 5A). Moreover,there is an extra TMH at the N-terminus of cyt. c 1 , which strongly binds to cyt. b and traps ap hospholipid molecule inside the complex. Therefore,t he cytochrome bc 1 complex from A. aeolicus is able to grab a1 ,4-naphthoquinone from the Q-pool and to sequester aphospholipid in the membrane ( Figure 7B), thus forming am ore stable conformation at high temperature and providing asuitable environment for the internal electron transfer reaction.
On the other hand, the electron transport pathway within the cytochrome bc 1 complex from A. aeolicus appears to be stabilized by enhanced binding of the prosthetic groups.Inthe cyt. b subunit of A. aeolicus,T yr38 and Arg119 bind to the two carboxyl groups of the cofactor heme b H ,t hen Glu254 and Arg222 bind to the two carbonyl groups of the 1,4-naphthoquinone substrate.Thus,both cofactor and substrate in the Q i reaction site are more stabilized in the cytochrome bc 1 complex of A. aeolicus,c ompared to the complexes from other species.A th igh temperatures,t he thermal motions of molecules are enhanced, so their relative positions and distances change quickly,w hich might reduce the electron transfer efficiency between them.  In aprevious study,the crystal structure of cytochrome c 555 from A. aeolicus was determined at 1.15 resolution. [15] Interestingly,t here is also au nique 14-residue long extra helix in this structure,w hich strongly binds to the core structure of c 555 .T his helix motif is demonstrated to contribute to the hyperstability of the c 555 ,and to help A. aeolicus to adapt to the hyperthermophilic environment. In our structure, the N-terminal extra helix TMH1 of cyt. c 1 is much longer than that of c 555 ,a nd the various hydrophobic residues of TMH1 increase the interface between the cyt. b and cyt. c 1 subunits by 60 % (Figure 6). This additional interaction could also contribute to the hyperstability of the overall complex. More importantly,T MH1 of cyt. c 1 fixes ap hospholipid molecule in au nique hydrophobic groove.T his kind of conformation has not been found in other mesophilic species, which could help A. aeolicus to adapt to the hyperthermophilic environment.
In the structure,w ef ound density for two 1,4-naphthoquinone substrates in the Q i site of cyt. b,a round Arg222 residues on TMH4 ( Figure 7A). Thet ail of the 1,4-naphthoquinone stretches towards the membrane core outside of the binding pocket without interacting with cyt. b. However, the tail of the ubiquinone identified in previous structures stretches towards heme b H . [3a] In addition, the entire antimycin Ai nhibitor was found in the Q i pocket and shows as trong interaction with Glu254. There are five hydrogen bonds seen between antimycin Aa nd cyt. b. In contrast, previously only the head group of this inhibitor was reported to be bound to the Q i site. [1d, 2c, 16] It is possible that this location represents the NQ channel from the Q-pool, in which the substrates are transferred into or out of the catalytic center ( Figure 7B). Theunique Arg222 residue helps to stabilize the substrate binding and may help the substrates to reach their binding site close to heme b H in the hyperthermophilic environment. Data collection and structure determination details are summarized in Table 1.

Conclusion
In summary,w es olved the 3.3 structure of respiratory complex III from the hyperthermophilic chemoautotrophic eproteobacterium Aquifex aeolicus and revealed the structural basis for the hyperstability of proteins in an extreme thermal environment. It is the first 1,4-naphthoquinone structure in the Q i sites.Several residues unique for thermophilic bacteria were detected that provide additional stabilization for ligand binding and for the structure of the whole complex. It was able to grab 1,4-naphthoquinones and to sequester phospholipids in the membrane,t hus forming am ore stable conformation at high temperature and providing as uitable environment for the internal electron transfer reaction. These results provide structural basis for the hyperstability of the cytochrome bc 1 complex in an extreme thermal environment.