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

Abstract The heme‐copper oxidase superfamily comprises cytochrome c and ubiquinol oxidases. These enzymes catalyze the transfer of electrons from different electron donors onto molecular oxygen. A B‐family cytochrome c oxidase from the hyperthermophilic bacterium Aquifex aeolicus was discovered previously to be able to use both cytochrome c and naphthoquinol as electron donors. Its molecular mechanism as well as the evolutionary significance are yet unknown. Here we solved its 3.4 Å resolution electron cryo‐microscopic structure and discovered a novel dimeric structure mediated by subunit I (CoxA2) that would be essential for naphthoquinol binding and oxidation. The unique structural features in both proton and oxygen pathways suggest an evolutionary adaptation of this oxidase to its hyperthermophilic environment. Our results add a new conceptual understanding of structural variation of cytochrome c oxidases in different species.


Introduction
In all respiring organisms electrochemical proton gradients drive the flux of protons back through the membrane via ATP-synthases,which produces adenosine-5'-triphosphate by attaching an inorganic phosphate to adenosine-5'-diphosphate.I na erobic organisms,t he electrochemical proton gradient is generated by as eries of proton translocation reactions in the respiratory chains.C ytochrome c oxidase (CcO) is the terminal enzyme in the respiratory chains of many aerobic organisms.Itislocated in the inner membrane of mitochondria and bacteria, and catalyzes the electron transfer from cytochrome c to molecular oxygen that is reduced to water. Studies on this integral membrane protein complex revealed that eight protons are taken up from the matrix side of mitochondrial membrane or from the bacterial cytoplasm (N-side), four protons are pumped across the membrane into the intermembrane space of mitochondria or the periplasm of gram-negative bacteria (P-side), while another four protons are used for water formation. [1] CcOi samember of the heme-and copper-containing terminal oxidases (HCOs) superfamily, [2] which also includes ubiquinol oxidases (QOXs),f or example,t he well-studied cytochrome bo 3 from Escherichia coli (E. coli) [3] but not the cytochrome bd oxidases from the same bacterium. [4] HCOs are classified into three families,A ,Band C, based on their amino acid sequences and proton transfer pathways. [5] They are multi-subunit complexes,f or example,t hey possess 14 protein subunits in mammalian mitochondria [6] and 3subunits in some bacteria. [7] Thec onserved central catalytic subunit Ic ontains two heme groups and ac opper atom (Cu B ). Thel ow-spin heme can be aheme a or aheme b in prokaryotes, [3,8] whereas only heme a has been found in mitochondrial cytochrome c oxidases. [9] Thel ow-spin heme a in the A-family CcOf rom Bos taurus (BtCcO) [9b] and heme b in the B-family CcOfrom Thermus thermophilus (TtCcO) accept electrons from Cu A , [7] and transfer them to the active site that is formed by the highspin heme a 3 and Cu B .The low-spin heme b in the QOX from E. coli directly accepts electrons from ubiquinol and transfers them to the binuclear center that is formed by ah igh-spin heme o 3 and Cu B . [10] When the binuclear center becomes doubly reduced, dioxygen binds to the heme iron and is reduced to water. Therequired protons are provided from the cytoplasmic side.
Subunit Io ft he CcOs most often contains 12 transmembrane helices (TMHs). An exception is TtCcOw hose subunit Ipossesses 13 transmembrane helices. [7] Subunit II is well conserved in the A-and B-families with its binuclear Cu A center located at the P-side and accepting electrons from cytochrome c, [11] whereas in QOXs subunit II contains two TMHs with Cu A absent. [10,12] Subunit III is present in mitochondrial and most bacterial HCOs in A-family,a nd could be fused to subunit I. [13] TtCcOisthe best studied HCO of the B-family.Its crystal structures in the oxidized state have been reported at resolutions of 2.4 [7] and 1.8 , [14] respectively.I ts proton pathway was found to be similar to the K-pathway of Afamily CcO. [15] Differently,c ompared to the A-family CcO (PdCcO) from Paracoccus denitrificans (P. denitrificans), subunit II of TtCcOo nly contains one TMH. Thep osition of the second N-terminal TMH of PdCcOs ubunit II is occupied by the additional subunit IIa of TtCcOi na n opposite orientation. [7] Although it has been challenged recently, [16] several studies suggested the efficiency of proton pumping in B-family CcOs (H + /e À = 0.5) [17] appears to be lower than that of A-famliy CcOs (H + /e À = 1). [18] Aquifex aeolicus (A. aeolicus)i sahyperthermophilic chemolithoautotrophic bacterium. Thecytochrome c oxidase from A. aeolicus,A aCcO, was previously discovered belonging to B-family HCO,a nd interestingly could use both cytochrome c and ubiquinol as electron donors, [19] which is aunique feature as amember of B-family HCO.Weoriginally hypothesized it would be caused by formation of supercomplex between AaCcOa nd complex III, providing additional quinol binding sites to enable its direct oxidation bypass cytochrome c. However,b ys olving the structure of A. aeolicus complex III, [20] we did not find novel structural features to support this hypothesis.I na ddition, 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] Thus,t he ubiquinol oxidation activity of the specimen would most likely come from AaCcOitself.Itwould be the own structural variation of AaCcOt og ain the function of additional quinol oxidation.
To gain insights into the molecular mechanism of AaCcO and also to understand how AaCcOa dapts its structure to keep stability and activity under hyperthermophilic growth conditions,w ep urified the AaCcOf rom native membranes and determined its structure at 3.4 resolution by using single-particle electron cryo-microscopy (cryo-EM). We found ad imeric form of AaCcOw ith an ovel binding site of the native quinol (VII-tetrahydromultiprenyl-1,4 naphthoquinone,N Q) at the dimeric interface,w hich could allow NQH 2 to be adirect electron donor bypassing cytochrome c. Further structure comparisons revealed structural variations of AaCcOtoincrease structural stability and alter the proton transfer pathway as well as the oxygen diffusion pathway for its adaptation of the hyperthermophilic growth environment.

Results
Overall structure of AaCcO dimer 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 3.4 according to the gold standard FSC 0.143 (Fourier Shell Correlation) criterion (Figures S2, S4 and S5;Movie S1). TheAaCcOdimer exhibits aC 2s ymmetry and contains three subunits ( Figure 1A), subunit I(CoxA2, 63.9 kDa) with the heme b and the heme a 3 / Cu B active site,s ubunit II (CoxB2, 16.8 kDa) with the Cu A center, and subunit IIa (5.2 kDa) ( Figure S1). It has dimensions of 84.7 in height and 107.7 in length. Thelength of the AaCcOmonomer is 55.0 .The cofactors Cu A ,Cu B ,heme a 3 and heme b are well resolved ( Figure 1B and Figure S4) with the edge-to-edge distance between Cu A and heme b Fe 15.4 ,and the edge-to-edge distances from heme b to heme

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Forschungsartikel a 3 and from heme a 3 to Cu B are 5.0 and 5.1 ,respectively. Theo verall structure of AaCcOi ss imilar to that of TtCcO with aroot mean square deviation (RMSD) of 1.02 for the aligned C a atoms.

Subunit I: CoxA2
Surprisingly,s ubunit I( CoxA2) contains 14 TMHs,t wo more than the canonical structures of CcOs.T he two additional TMHs of CoxA2 are found at the C-terminus (Figure 2A). This observation is consistent with sequence alignments which show subunit Ii np rokaryotes has al onger-Cterminus than that in eukaryotes ( Figure S3). Thee xtra two TMHs bind to the outer surface of TMH5, TMH6, TMH7, and TMH8 of CoxA2. Asuperimposition of the AaCcOa nd TtCcOs tructures shows that the location of the 13 th TMH is same in both complexes ( Figure 2B). Thes tructural superimposition also shows that the additional TMH14 occupies nearly the same site of one TMH of subunit III of the aa 3 -type CcO( Figure 2C). Theloops connecting the TMHs of CoxA2 are relatively short and this observation is in accordance with the typical properties of thermostable proteins. [22] Interestingly,the loop between TMH8 and TMH9 at the cytoplasmic surface is longer than that of BtCcO. This loop points to the dimer interface ( Figure 2D).

Subunit II:C oxB2 and Subunit III:IIa
Subunit II (CoxB2) contains one TMH and aten-stranded b-barrel ( Figure 3A). The b-barrel forms apolar domain that is located at the periplasmic side.The binuclear Cu A center is bound by the conserved residues His96, His139, Cys131, and Cys135. Thedistance between the two copper atoms is 2.7 . Thec onserved residues Tr p121 and Ty r122 in P. denitrificans were proposed to play important roles in the electron transfer from cytochrome c to Cu A . [11] These two conserved residues are also observed in CoxB2 as Tr p66 and Ty r67 (Figure-s3A,B).
Subunit IIa of AaCcOh as been identified previously, [19] and the corresponding density was found and traced in our structure.Itcontains only one TMH that possesses alocation identical to that of the first TMH of subunit II in PdCcObut with opposite orientation ( Figure 3A). Subunit IIa is involved in the formation of the dimer interface and interacts with lipids and quinone ( Figure 3C).

The AaCcO dimer
Ac omparison of the AaCcOd imer structure with the BtCcOd imer structure (PDB entry 2OCC) shows that their dimer interfaces are completely different ( Figure 4A). The AaCcOd imer is formed via interactions of its major subunit CoxA2 while this is not the case for BtCcO( Figure 4B). Few protein-protein but fruitful protein-lipid interactions are observed in the dimeric interface.S trong hydrogen bond networks between protomers are observed among residues Ty r328, Arg337 and Glu339 at the loop region between TMH8 and TMH9 ( Figures 4C,D). Ah ydrophobic cavity is  found in the interface near the cytoplasmic side,w hich is occupied by many lipid molecules ( Figure S4). TwoP Es (phosphatidylethanolamine) and two PGs (phosphatidylglycerol) lipid molecules are identified in the cavity ( Figure 4C). And four more PGs are found at the vicinity ( Figure 4C). Interestingly,t wo quinol molecules (NQ) are found at the interface with the head group orientation towards the P-side ( Figure 4C). Our subsequent lipidomics mass spectrometry analysis of co-purified lipids in the sample confirmed the exact chemical composition of PE and PG and the mass spectrometry analysis of native A. aeolicus membranes identified the native quinol molecule as VII-tetrahydromultiprenyl-1,4-naphthoquinone [23] (Figure S6).

The NQ binding site
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). Thus,o xidation of NQH 2 most likely occurs in the AaCcOitself,which can be proved by the dimeric structure of AaCcOa nd the existence of NQ molecules at the dimer interface ( Figure S4). Each NQ molecule is deeply buried in the hydrophobic groove formed by subunits IIa and coxA2 ( Figure 5A). Many hydrophobic residues interact with the NQ aliphatic chain, including Va l36, Ile33, Met32, Leu29, Phe25, and Phe21 of IIa and Phe430, Met437, Va l438, Va l441 in TMH11 of CoxA2. In particular, one carbonyl oxygen of NQ is found to bind to Glu39 of subunit IIa (Figure 5A), aresidue presents only in A. aeolicus ( Figure S7A). Ad eprotonated Glu39 would form as trong hydrogen bond with the hydroxyl group of NQH 2 and accept one proton upon oxidation of NQH 2 .T obenoted, the NQ tail is buried inside one protomer while its carbonyl oxygen is proximal to heme b of another protomer ( Figures 5A,B). The edge-to-edge distance between NQ and heme b is 15.0 . With this distance direct electron transfer is possible.I n addition, the existence of several aromatic residues (Phe37, Tr y38, Ty r53, and Phe445) would be also possible involved in the electron transfer from NQH 2 to heme b ( Figure 5B). This quinol binding pocket is similar to the menaquinol binding pocket in cytochrome aa 3 -600 menaquinol oxidase from Bacillus subtilis ( Figure S7B). [24] Only aKproton pathway exists in AaCcO Based on the structure of aB-family CcO, the presence of three possible proton pathways,n amed K-, D-, and Qpathways,w ere previously suggested. [7] Ther esidues for forming these pathways are usually conserved between different species with only al imited number of mutations. TheK -pathway,n amed after its essential lysine residue Lys354, was identified previously. [25] Structural superposition of the crystal structure of TtCcOa nd the cryo-EM structure of AaCcOreveals the presence of the K-pathway in AaCcO, except that at the start Glu516 in the subunit Io fT tCcOi s mutated to His515 in AaCcO( Figure 6A). In this pathway, protons can be transferred from His515 and Asp516 at the N side,v ia Ser252, Ty r237, Thr303, Ty r233, Ser300, and Tyr226 to the active site that is formed by the high spin heme a 3 and Cu B .I nt he classical K-pathway,t here is usually ac onserved Glu of subunit II as apotential proton entry point, [26] which is Glu15 in the subunit II of TtCcOa nd also conserved in AaCcO(Glu 5o fs ubunit II, Figure S8).
Structural superposition also reveals the potential D-and Q-pathway of AaCcO ( Figures 6B,C). In the D-pathway of TtCcO, protons can be transferred from Glu17 on the cytoplasmic side,v ia Ty r91, Ser109, Ser155, Thr156, Ser197, Thr231, and several water molecules,t ot he heme a 3 active

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Forschungsartikel site.However,inAaCcO, the entrance for protons is blocked by several hydrophobic residues,i ncluding Ala82, Leu89, Ala79, and Ile10. Furthermore,r eplacing hydrophilic resides to hydrophobic ones Va l74, Ile186, and Phe220 does not allow proton transfer ( Figure 6B). As imilar situation was also found for the potential Q-pathway of AaCcO( Figure 6C). Thus,o nly the K-pathway does exist for proton transfer in AaCcO.

An unobstructed oxygen diffusion pathway in AaCcO
Based on the crystal structure of TtCcO, the presence of aY -shaped oxygen diffusion pathway with two entry points was suggested [7] (Figure 7A). Interestingly,inAaCcOone Vshaped potential oxygen diffusion pathway is observed (Figure 7B). There is only one entry point to this diffusion pathway,which starts at the middle of the membrane.Inthis pathway,O 2 enters ah ydrophobic gate formed by Ile194, Ile193, Va l138, and Leu135, turning at Phe220, Phe123, Va l65, Ile66, and Tr y121, then passes near Trp228, Va l224, Va l225, Phe220, Phe217, Tr p218 to reach the heme a 3 active center ( Figure 7B). Sequence alignment shows that most of the residues lining the putative oxygen pathway are conserved, except Phe113 in AaCcO( Figure S9). As tructural superposition of TtCcOand AaCcOreveals that the oxygen entry point 2m ight be blocked by Phe113 in AaCcO( Figure 7C).

Discussion
Thes tructures and functions of respiratory complexes from different species have been extensively studied in past years. [28] Compared to conventional cytochrome c oxidases that use cytochrome c as the electron donor,A aCcOc an directly oxidize quinol substrates besides cytochrome c. [19] In the present study,weexplored the high-resolution structure of AaCcOb ysingle particle cryo-EM and got insights into the molecular mechanism of how AaCcOc ould use both cytochrome c and quinol as electron donors by discovering the existence of the native quinol molecules NQs bound at the dimeric interface.T he edge-to-edge distance from NQ to heme b is close enough to enable ad irect electron transfer from NQH 2 to the active binuclear center via heme b. The proximal aromatic residues between NQ and heme b would presumably enhance the rate of electron transfer. Subunit IIa was found to be important for NQ binding by ligation of the head group of NQ and the residue Glu39 of Subunit IIa presumably plays ar ole of stabilizing NQ during electron transfer. Notably,s uch electron transfer could only happen between NQ bound to one protomer and heme b of another protomer. Thus,the dimerization of AaCcOnot only provides an ew interface for NQ binding but also be necessary for direct electron transfer from NQ.A ny regulation factor including thermal fluctuation that alters the formation of AaCcOd imer might affect its activity of direct NQH 2 oxidation, which could likely explain the low quinol oxidation activity measured previously. [19]   The oxygen channels were calculated and predicted using MOLE2. [27] Previous studies found BtCcOt of orm ah omodimer in the crystals [29] while it appears as am onomer in all supercomplex structures. [30] Ar ecent cryo-EM study discovered another intact 14 th subunit (NDUFA4) of human cytochrome c oxidase,which is important to keep it in amonomeric active form but was absent in the previous dimeric less active form. [31] At the same time,t he structure of an active monomeric form of BtCcO was also determined by X-ray crystallography. [32] Our present work does not rule out the existence of as upercomplex in A. aeolicus,w hich has been suggested in aprevious study. [33] However,the insensitivity of ubiquinol oxidation activity of the potential supercomplex to stigmatellin indicated that AaCcOi tself has the activity of ubiquinol oxidation. [19] After solving the structure of AaCcO, its ubiquinol oxidation activity could be only explained by its dimeric form. Furthermore,t his dimeric form is different from that of all other reported CcOd imers.W ealsos uperimposed the dimeric structure of AaCcOinto other reported respiratory supercomplexes from Mycolicibacterium smegmatis (M. smegmatis), [34] Saccharomyces cerevisiae (S. cerevisiae) [28e] and Sus scrofa (S. scrofa), [35] and found the dimeric interface of AaCcOd oes not overlap the interface between complex III and complex IV in the supercomplexes from M. smegmatis and S. scrofa ( Figure S10). Thus,t of orm as upercomplex, such dimeric form of AaCcOwould not need to be broken.
Considering the hyperthermophilic growth environment of A. aeolicus,itwould be interesting to investigate the unique structural features of its respiratory chain complex and understand the mechanism of structural adaptation suitable for hyperthermophilic environment. 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,w e also found that subunit IofAaCcOp ossesses two additional C-terminal transmembrane helices,T HM13 and TMH14, in comparison with eukaryotic CcOs,o rs till one additional TMH when comparing with TtCcO, at hermophilic prokaryotic CcO. Thus,i tm ight be possible that the presence of the extra TMHs of AaCcOenhances its thermal stability suitable for the hyperthermophilic growth conditions.Inaddition, the membrane-anchored cytochrome c 555 might bind to this TMH14, as proposed for the aa 3 -type CcOf rom P. denitrificans. [36] Based on the crystal structure of TtCcO, three proton transfer pathways (K, D, and Q) were proposed. [7] Mutations of critical residues on D-pathway (S109A) and Q-pathway (T396V) showed little influence on the enzymatic activity. [15,37] Structural superposition of TtCcOand AaCcOreveals that the D-and Q-proton pathways in AaCcOa re both blocked by multiple hydrophobic residues.T herefore,even if the D-and Q-proton pathways in TtCcOw ere active,t he same pathways in AaCcOshould be closed and inactive.Only the K-pathway appears to be present in AaCcO. Besides the proton transfer pathway,t he potential oxygen diffusion channel of AaCcOa lso varies in comparison with that of TtCcO. Along with conserved oxygen channels being suggested for CcOs from Rhodobacter sphaeroides, [38] P. dentrificans [39] and B. taurus, [9b] aY -shaped oxygen channel was also reported in TtCcO, suggesting that there are two entry points for oxygen. However,structural superposition of AaCcOand TtCcOs uggest that only one oxygen diffusion channel exists and forms aV -shape in AaCcO. Thes econd oxygen entry point 2f ound in TtCcOi sb locked by residue Phe113 in AaCcOa tt he equivalent position. Thel arger the void in aprotein, the smaller is its stability. [40] Theadapted structure of AaCcOwith only the Kproton pathway present and the Vshaped unobstructed oxygen channel with more hydrophobic residues blocking one entry appears to be evolutionary advantageous to keep the balance between its enzymatic activity and structural stability in the hyperthermophilic environment.

Conclusion
In summary,wesolved the 3.4 structure of cytochrome c oxidase from the hyperthermophilic bacterium Aquifex aeolicus,revealed the molecular mechanism that this oxidase uses both cytochrome c and quinol as electron donors,m ade structural insights into its thermal stability,a nd suggested an evolutionary adaptation of this oxidase to keep the balance between its enzymatic activity and structural stability for the hyperthermophilic growth condition. These results provide structural basis for molecular mechanism and the evolutionary significance of cytochrome c oxidases in an extreme thermal environment.

Data and materials availability
Thea tomic coordinates of the cytochrome c oxidase of Aquifex aeolicus reported in this paper have been deposited in Worldwide Protein Data Bank (PDB) (http://www.rcsb. org) with the accession codes 7DEG.The corresponding maps have been deposited in the Electron Microscope Data Bank (EMDB) (http://emdatabank.org) with the accession codes EMD-30657.
Natural Science Foundation of China (31830020). This work was also supported by grants from Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft (Cluster of Excellence Macromolecular Complexes,F rankfurt) and the Ministry of Science and Te chnology of China (2018YFA0901102 and 2019YFA0904101). Open access funding enabled and organized by Projekt DEAL.

Conflict of interest
Theauthors declare no conflict of interest.