Structure of the cytochrome aa3-600 heme-copper menaquinol oxidase bound to inhibitor HQNO shows TM0 is part of the quinol

Jingjing Xua,1,2, Ziqiao Dingb,1, Bing Liua,1,2, Sophia M. Yib,3, Jiao Lia,2, Zhengguang Zhanga,2, Yuchen Liua,2, Jin Lic, Liu Liuc, Aiwu Zhoud, Robert B. Gennisb, and Jiapeng Zhua,e,2,4

aSchool of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 210023 Nanjing, China; bDepartment of Biochemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801; cDivision of Medicinal Chemistry, PharmaBlock Sciences (Nanjing), Inc., 211800 Nanjing, China; dDepartment of Pathophysiology, Shanghai Jiao Tong University School of Medicine, 200025 Shanghai, China; and eJiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, 210023 Nanjing, China

Edited by Hartmut Michel, Max Planck Institute for Biophysics, Frankfurt, Germany, and approved December 4, 2019 (received for review August 30, 2019)

Virtually all proton-pumping terminal respiratory oxygen reductases cytochrome c binding domain and the CuA binding motif in sub- are members of the heme-copper superfamily. Most unit II. In addition, subunit I of all of the quinol oxidases contains of these use reduced cytochrome c as a source of electrons, 15 TM helices rather than the canonical 12 TM spans (1). These but a group of enzymes have evolved to directly oxidize membrane- extra membrane-spanning helices are designated as TM0 at the bound quinols, usually menaquinol or ubiquinol. All of the quinol N terminus and TM13 and TM14 at the C terminus of subunit oxidases have an additional transmembrane helix (TM0) in subunit I I. The presence of TM0 is unique to the quinol oxidases, while that is not present in the related cytochrome c oxidases. The current TM13 and TM14 are found in some cytochrome c oxidases, e.g., work reports the 3.6-Å-resolution X-ray structure of the cytochrome cytochrome caa3 from Bacillus subtilis (7). aa3-600 menaquinol oxidase from Bacillus subtilis containing 1 While there are numerous structures of cytochrome c oxidases equivalent of menaquinone. The structure shows that TM0 forms from a variety of species (2, 8–12), there is only 1 structure part of a cleft to accommodate the menaquinol-7 substrate. Crystals reported for a heme-copper quinol oxidase, E. coli cytochrome which have been soaked with the quinol-analog inhibitor HQNO bo3 ubiquinol oxidase (13). This contains 4 subunits, 3 (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single of which are homologous to the 3 mitochondrial encoded subunits CHEMISTRY binding site where the inhibitor forms hydrogen bonds to of the eukaryotic cytochrome c oxidases (14). The structure of residues shown previously by spectroscopic methods to interact with cytochrome bo3 is missing ∼25% of the expected amino acid res- the semiquinone state of menaquinone, a catalytic intermediate. idues, including TM0, TM13, and TM14 in subunit I, and does not

heme-copper oxidoreductase | | proton pumping Significance

he mitochondrial aerobic respiratory chains of virtually all The proton-pumping quinol oxidases, close orthologs of the BIOCHEMISTRY Taerobic eukaryotes terminate with a proton-pumping heme- cytochrome c oxidases, directly oxidize membrane-bound copper oxygen reductase, cytochrome aa3,whichisanaa3-type quinols to reduce oxygen to water. As one of the major con- (1). In mammals, the enzyme contains 13 tributors of proton-motive force, they exist widely in , subunits, 3 of which (subunits I, II, and III) are encoded in the including many pathogens. Here, we describe the structure of a mitochondrion and 10 in the nucleus (2). Most aerobic prokaryotic quinol oxidase that clearly defines the protein features that organisms also contain a proton-pumping heme-copper oxygen have evolved to allow oxidation of a membrane-bound quinol reductase as part of the respiratory chain, but these enzymes as a substrate. TM0, a transmembrane helix in subunit I that is typically contain fewer subunits (2 to 4) (1, 3). All heme-copper not present in the related cytochrome c oxidases, forms part of oxygen reductases contain a homolog of subunit I, which usually a cleft that would accommodate the menaquinol-7 substrate. contains 12 transmembrane (TM) helices, along with the binding Cocrystal structure with the quinol-analog inhibitor HQNO sites for a low-spin heme (e.g., heme a) and the high-spin heme- (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveals copper binuclear center (e.g., heme a3-CuB). The subscript “3” a single binding site where the catalytic intermediate binds. designates the heme where O2 binds and is reduced to water. Subunit I contains the proton-conducting channels which facilitate Author contributions: R.B.G. and J.Z. designed research; J.X., Z.D., B.L., S.M.Y., Jiao Li, Z.Z., the translocation of protons from the electrically negative side of Y.L., Jin Li, L.L., and J.Z. performed research; Jin Li and L.L. contributed new reagents/ analytic tools; Z.D., B.L., A.Z., R.B.G., and J.Z. analyzed data; and J.X., Z.D., B.L., S.M.Y., Jiao the membrane either to the for the chemical conversion Li, A.Z., R.B.G., and J.Z. wrote the paper. of O2 to 2 H2O or to the positive side of the membrane, i.e., The authors declare no competing interest. pumped protons (4, 5). Prokaryotic heme-copper oxygen reduc- This article is a PNAS Direct Submission. tases, besides having fewer subunits than the eukaryotic homologs, Published under the PNAS license. also can utilize different hemes (3, 6). Whereas the mitochondrial Data deposition: The coordinates for the model of cytochrome aa3-600 from B. subtilis enzyme contains 2 equivalents of heme A (a chemical modification have been deposited in the Protein Data Bank, https://www.rcsb.org (PDB ID codes 6KOB, of heme B), prokaryotic heme-copper oxygen reductases can uti- 6KOC, and 6KOE). lize different combinations of heme B, heme O, and heme A. For 1J.X., Z.D., and B.L. contributed equally to this work. bo example, cytochrome 3 from contains 1 equiv- 2Present address: School of Medicine and Holistic Integrative Medicine, Nanjing University alent of heme B and 1 equivalent of heme O, which binds to O2. of Chinese Medicine, Nanjing 210023, China. Most prokaryotic heme-copper oxygen reductases (A, B, and 3Present address: Department of Molecular Biophysics and Biochemistry, Yale University, C families) (1) use reduced cytochrome c as a substrate, but a New Haven, CT 06511. subfamily of the A family utilizes a membrane-bound quinol, 4To whom correspondence may be addressed. Email: [email protected]. usually ubiquinol or menaquinol, as a substrate in place of cy- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ tochrome c. The quinol oxidases can be distinguished from the doi:10.1073/pnas.1915013117/-/DCSupplemental. homologous cytochrome c oxidases by the absence of both the

www.pnas.org/cgi/doi/10.1073/pnas.1915013117 PNAS Latest Articles | 1of5 Downloaded by guest on September 28, 2021 contain ubiquinone. Despite these deficiencies, the structure did reveal a cluster of amino acid residues in subunit I that are con- served among ubiquinol oxidases (R71I,D75I,H98I, and Q101I; E. coli numbering, and the superscript I designates subunit I), sug- gesting that these might constitute a portion of an ubiquinol binding site (13). Mutagenesis showed their functional importance, and pulsed electron paramagnetic resonance (EPR) studies have clearly demonstrated that R71I,D75I,H98I,and,toalesserextent, Q101I directly interact with and stabilize the ubisemiquinone (SQ-•) catalytic intermediate (15–19). In the current work, the X-ray structure of the cytochrome aa3-600 proton-pumping menaquinol oxidase from B. subtilis (20–22) is reported at 3.6-Å resolution. This enzyme contains 4 subunits and is a close ortholog of E. coli cytochrome bo3. The sequences of subunit I of these 2 enzymes are 50% identical, even though the enzymes use different quinol substrates and contain different hemes. Cytochrome aa3-600 contains 2 equiva- lents of heme A in place of 1 equivalent each of heme B and heme O found in cytochrome bo3.TheB. subtilis and E. coli enzymes utilize menaquinol-7 and ubiquinol-8, respectively. The “600” in the name of the menaquinol oxidase designates the peak of the alpha absorption band due to heme a. In contrast to the structure Fig. 1. Surface representations (A and B) and cartoon representations (C bo aa and D) of the structure of cytochrome aa3-600 from B. subtilis. The four of cytochrome 3, cytochrome 3-600 is crystallized with 1 subunits are color-coded: subunit I (blue), subunit II (red), subunit III (gray), equivalent of the native substrate (menaquinol-7). Importantly, and subunit IV (brown). TM0 has no counterpart in any of the cytochrome c TM0 was resolved and appeared to form part of a cleft facing the oxidases and is unique to the heme-copper quinol oxidases. lipid bilayer into which the hydrophobic poly-isoprene side chain of the substrate, menaquinol-7, can fit while being oxidized. In addition, structures have also been determined with 1 equivalent cytochrome c oxidase from Thermus thermophilus (PDB ID code of the inhibitors HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 2YEV), but different from the aa3-type cytochrome c oxidase from 3-iodo-HQNO bound to the protein. The inhibitor is hydrogen R. sphaeroides (PDB ID code 1M56). The farnesyl tails of heme a3 I I I bonded to residues R70 ,D74, and H94 (B. subtilis numbering), and heme o3 from cytochrome aa3-600 and cytochrome bo3,re- which have been shown spectroscopically to stabilize the semi- spectively, have similar orientations. quinone state of the menaquinol substrate (21, 22). The 2 proton-conducting channels, named the D channel and the K channel, that are present in A-family heme-copper oxygen Results reductases (23) are also present in cytochrome aa3-600, which is Additional TM Helices in Subunit I Compared to the Canonical also in the A family. The D channel starts from a highly conserved I Cytochrome c Oxidases. Whereas subunit I of most cytochrome c aspartate residue, D131 , at the entrance of the channel; includes I I I I I I oxidases contains 12 TM helices, subunit I of both cytochrome bo3 polar residues T207 , N138 , N120 , N203 ,S141,T200,and I I and aa3-600 (and all quinol oxidases) contains 15 TM helices; 1 T197 ; and ends at E282 , which is the branching point of chemical additional TM span, TM0, at the N terminus; and 2 additional TM protons and pumped protons, as determined from previous stud- spans, TM13 and TM14, at the C terminus. Although not resolved ies. The K channel features a conserved lysine residue K358I and I I I in the crystal structure of cytochrome bo3 (Protein Data Bank contains a series of polar residues S295 , T355 , and Y284 .These [PDB] ID code 1FFT), these 3 helices are confidently modeled in key residues in both channels exist at the same places as in the the structure of cytochrome aa3-600 (Fig. 1). The superposition of cytochrome bo3 quinol oxidase (SI Appendix,Fig.S3). the cytochrome aa3-600 menaquinol oxidase structure and the Subunit II. Unlike cytochrome c oxidases, there is no CuA-binding c structure of the aa3-type cytochrome c oxidase from Rhodobacter site or cytochrome docking site in subunit II of quinol oxidases. sphaeroides (PDB ID code 1M56) shows that TM13 and TM14 Whereas subunit I of B. subtilis cytochrome aa3-600 subunit I has overlap with the first 2 helices of subunit III of the R. sphaeroides ∼50% amino acid sequence identity with that of the E. coli cyto- enzyme. TM0 has no counterpart in any of the cytochrome c ox- chrome bo3, the sequences of subunit II have only 31% identity. idases and is unique to the heme-copper quinol oxidases. Subunits III and IV. Subunits III of cytochrome aa3-600 and cyto- Significantly, the side chains of TM0 are not immediately chrome bo3 are 44% identical, and in both enzymes, subunit III adjacent to the nearby TM helices, but form a groove along with has 5 TM helices. The positions of these 5 helices overlap with TM1 and TM2 which appears to be well placed to facilitate the helices TM3 to TM7 in canonical cytochrome c oxidases (e.g., binding of menaquinol-7 from the membrane bilayer (Fig. 2). from R. sphaeroides PDB ID code 1M56). As previously noted, The residues that are hydrogen bonded to the menasemiquinone TM13 and TM14 from subunit I of cytochrome aa3-600 overlap species, R70I, D74I, and H94I, are located at the “top” of TM1 with TM1 and TM2 of subunit III of canonical cytochrome c and TM2 (external face of the membrane). Two consecutive lysine oxidases, suggesting an evolutionary “shift” of these TM helices residues on the cytoplasmic side of TM0, K38I, and K39I are in from the N terminus of subunit III in the canonical cytochrome good positions to interact with the phospholipids to anchor TM0 c oxidases to the C terminus of subunit I. The gene encoding to the membrane. subunit III is immediately downstream from that encoding subunit I in most organisms. Similar Features of Cytochrome aa3-600 and Cytochrome bo3. The function of subunit IV is not known, and this subunit is the Subunit I. The structures of the binuclear center and the porphyrin least conserved, with 29% identity between cytochrome aa3-600 and rings of the high- and low-spin hemes are essentially identical for cytochrome bo3. The side chains of subunit IV are not resolved in both cytochrome bo3 and cytochrome aa3-600, apart from the the structure of cytochrome bo3. Subunit IV of cytochrome aa3-600 chemical modifications differentiating heme B, heme O, and contains 3 TM helices, as also modeled for cytochrome bo3 (13). heme A. The farnesyl tail of heme a, which is not present in Bound menaquinone-7. Cytochrome aa3-600 oxidase was solubilized, cytochrome bo3, has a similar orientation as in the caa3-type isolated, and crystallized by using the detergent dodecylmaltoside

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.1915013117 Xu et al. Downloaded by guest on September 28, 2021 detergent DDM. Hence, menaquinone-7 does not require any interaction with R70I, D74I, or H94I to remain bound to the enzyme in the presence of DDM. The mutations of these active-site residues also make cytochrome aa3-600 less sensitive to inhibition by HQNO. For example, in the presence of 30 μM HQNO, R70IH, H94ID, H94IF, and E97IQ have substantial residual activity: 8%, 45%, 64%, and 20%, re- spectively. These results suggest that these residues are at or near the binding site for HQNO, similar to the observations made with cytochrome bo3 (26). The structure of cytochrome aa3-600 with the Fig. 2. Side view (A)andtopview(B) of TM0, TM1, and TM2 of subunit I of 3-iodo-HQNO derivative of HQNO confirms that the inhibitor cytochrome aa3-600, showing the conserved cluster of residues that are con- I I I served in all heme-copper menaquinol oxidases, R70I, D74I, H94I, and E97II. binds directly to R70 ,D74,andH94, as discussed below. The groove lined by TM0, TM1, and TM2 forms the binding site for mena- Binding site for inhibitors HQNO and 3-iodo-HQNO. After soaking the quinol-7 within the membrane bilayer. Two consecutive lysine residues, K38I crystals with HQNO, the initial Fo − Fc map showed extra density and K39I, are proposed to interact with phospholipids in the inner leaf of near residues R70I, D74I, and H94I (Fig. 4A). However, due to the membrane bilayer. the limited resolution, it was not possible to locate the exact po- sition of individual atoms of the HQNO molecule. To better un- derstand how the inhibitor interacts with cytochrome aa3-600, (DDM). With this detergent, the enzyme was copurified with 1 3-iodo-HQNO was synthesized to take advantage of the strong equivalent of menaquinone-7 (22), the oxidized form of the en- dogenous substrate. In the omit map calculated by using a ligand- electron density of the iodine atom. After soaking the crystals with 3-iodo-HQNO, the initial Fo − Fc map clearly showed a positive free model, electron density was observed in the groove defined by σ TM0, TM1, TM2, and TM3 of subunit I. The electron density was peak stronger than 7 representing the position of the iodine σ insufficient to confidently fit a molecular model. Assuming this atom. At 3 , the density of the rest of the inhibitor analog was also visible (Fig. 4B). The 3-iodo-HQNO was hydrogen bonded to density represents the bound menaquinol-7, the electron density I I I I could be modeled with the menaquinol head group plus 3 of the 7 R70 ,D74, and H94 .H94 has well-defined electron density and isoprene units of the hydrophobic tail. The menaquinone head underwent a significant conformational change compared with the groupinthismodelwasnearV73I (TM1, subunit I) and F156I native enzyme (Fig. 3B). All heme-copper menaquinol oxidases I CHEMISTRY (TM4, subunit I), and the isoprenyl tail established hydrophobic have a conserved glutamate (E97 ) in TM2 (subunit I), near the I interactions with the hydrophobic residues in TM3 and TM0 (Fig. bound 3-iodo-HQNO, but the electron density of E97 is not well 3A). Higher-resolution data will be required to confirm this in- defined, suggesting conformational flexibility of this side chain. terpretation of the observed electron density. It cannot be ruled The residual electron density that was fit to a portion of out that this density is due to a detergent or lipid molecule. menaquinol-7 in the crystal of the native enzyme in the absence Site-directed mutagenesis and inhibition by HQNO. HQNO is a potent of the inhibitor was still observed in the presence of either inhibitor of cytochrome bo3 (24) and is generally considered to HQNO or 3-iodo-HQNO.

mimic the semiquinone state of both menaquinone and ubiqui- BIOCHEMISTRY none. Wild-type cytochrome aa3-600 was completely inhibited in Discussion the presence of 1 μM HQNO. In addition, similar to what was Proton-pumping heme-copper oxygen reductases that directly found with cytochrome bo3 (24), the formation of the menase- oxidize membrane-bound quinols are present in the aerobic miquinone by cytochrome aa3-600 was abolished in the presence respiratory chains of many bacterial species. By far, the best of 1 μM HQNO. This is shown in SI Appendix, Fig. S2. characterized is the cytochrome bo3 ubiquinol oxidase from E. The residues that interact with and stabilize the menasemiqui- coli (26, 27), and the current work presents the X-ray structure none in cytochrome aa3-600 have been identified both by analogy of a very close ortholog, the cytochrome aa3-600 menaquinol to cytochrome bo3 (24), but also by pulsed EPR methods (21, 22): oxidase from B. subtilis (22). Although the hemes and sub- I I I I R70 ,D74,andH94 (Fig. 2). It was reported (21) that the R70 H strates are different for these 2 enzymes, the sequences of mutant has a perturbed interaction with the menasemiquinone subunit I, which contains the binding sites for the hemes and species, but does not eliminate the EPR signal of the radical, as the catalytic sites, are 50% identical. does the equivalent mutation in cytochrome bo3 (25). Further- more, the R70IH mutant is more active than wild-type cytochrome aa3-600, indicating a greater plasticity or tolerance to structural perturbation for stabilization of the menasemiquinone species compared to cytochrome bo3. To further confirm the identity of the residues that interact with the menasemiquinone species in cytochrome aa3-600, additional mutations were generated, and the effects on the steady-state activities with both DMNH2 and with menadiol are shown in Table 1. The mutations of residues D74I and H94I were the most potent in reducing the activity of the enzyme, though the H94IF mutant retained 62% of the activity with DMNH2 as the substrate. The results strongly suggest that D74I and H94I interact directly with one of the menasemiquinone species and contribute to stabilizing the menasemiquinone, but that stabilization of the menasemiquinone is less sensitive to changes in the surrounding amino acid residues than the stabili- bo Fig. 3. (A) Menaquinone molecule in the groove. The menaquinone head zation of the ubisemiquinone species by cytochrome 3 (25). In group is near V73I and F156I.(B) Superposition of the native and inhibitor- addition, similar to the results obtained with cytochrome bo3 (25), complexed structures. Green, native; magenta, cytochrome aa3-600 com- each of the mutants shown in Table 1 is bound to 1 equivalent plexed with 3-iodo-HQNO. The maps are omit maps, calculated by using of menaquinone-7 after solubilization and purification in the models without the substrate and contoured at 1.0 σ.

Xu et al. PNAS Latest Articles | 3of5 Downloaded by guest on September 28, 2021 Table 1. Activities of the wild-type and mutant variants of quinol from the membrane bilayer; and 2) the addition of the cytochrome aa3-600 using soluble menaquinol analogs cluster of residues at the “top” of TM1 and TM2 that stabilizes − Enzyme turnover, s 1 (%) the semiquinone state of the quinone, a required catalytic intermediate.

Enzyme With DMNH2 (50 μM) With menadione (130 μM) Experimental Methods WT aa3-600 115 (100) 33 (100) Purification of Cytochrome aa3-600 Menaquinol Oxidase. The construct con- R70H* 177 (154) 51 (155) taining cytochrome aa3-600 genes, qoxABCD, was generated by PCR from the E97Q 192 (167) 12 (36) B. subtilis genome, and a hexahistidine tag was added at the C terminus of H94F 71 (62) 24 (73) qoxA. The gene was subcloned into the pDR111 plasmid (28) and eventually H94D 18 (16) 2 (6) integrated onto the genome of B. subtilis PY79 strain via homologous re- D74H 6 (5) 0 (0) combination (both the pDR111 plasmid and PY79 strain were gifts from D74N 6 (5) 6 (18) Yunrong Chai, College of Science, Northeastern University, Boston). B. subtilis PY79 cells containing the incorporated construct were grown in Luria–Bertani *Results reported in ref. 21. medium, collected, and resuspended in 50 mM potassium phosphate buffer (pH 8.0), with the addition of phenylmethylsulfonyl fluoride (Sigma), 10 mM (ethylenedinitrilo)tetraacetic acid, and DNase I (Sigma). Homogenized B. The 2 most important conclusions from the current work subtilis cells were disrupted by using a high-pressure homogenizer (ATS En- are that gineering), followed by a 10-min centrifugation at 20,000 × g. The membrane fraction was separated after 3-h ultracentrifuge at 180,000 × g. The isolated 1) TM0, the N-terminal TM helix in subunit I that is found only membranes were resuspended in 50 mM potassium phosphate buffer (pH 8.0) in heme-copper oxygen reductases that utilize quinol sub- and solubilized with 1% DDM on ice for 1 h. Insoluble material was removed strates, is at a location where it can be reasonably assumed by centrifugation at 180,000 × g for 30 min. The solubilized fraction was to form part of the binding site for the membrane-bound loaded onto an Ni-nitriloacetic acid (NTA) (Qiagen) column preequilibrated quinol substrates. This substrate is menaquinol-7 in the case with 50 mM potassium phosphate buffer and 0.1% DDM (pH 8.0). A gradient wash was applied with increasing concentration of imidazole (5 to 25 mM) in of cytochrome aa3-600. The groove facing the lipid bilayer that is formed by portions of TM0, TM1, TM2, and TM3 is the same buffer in order to remove nonspecific bound protein. His-tagged cytochrome aa3-600 was eluted with 50 mM imidazole in 50 mM potassium located just below the cluster of residues known to stabilize I phosphate buffer and 0.1% DDM (pH 8.0). The peak fractions from the Ni-NTA the semiquinone state of the menaquinol-7 substrate, R70 , column were pooled and concentrated 5-fold to ∼200 μL by using a 100-kDa I I D74 , and H94 (21, 22). These residues are located within molecular-mass-cutoff Vivaspin centrifugal concentrator. For subsequent TM1 and TM2. crystallization, the protein was twice diluted with 5 mL of buffer containing 2) The semiquinone-mimic HQNO that inhibits cytochrome 20 mM Tris·HCl (pH 7.8) and 50 mM NaCl and concentrated. In the presence of aa3-600, binds stoichiometrically to the enzyme and is hydro- aa3-600, DDM micelles could not pass through the 100-kDa cutoff concen- gen bonded to the 3 residues known to stabilize the semi- trator, and no detergent was needed in the exchanged buffer. The ultraviolet- quinone form of menaquinone-7 bound to the protein, R70I, vis spectra of the isolated enzyme, along with the sodium dodecyl sulfate/ D74I, and H94I (21, 22). Notably, H94I undergoes a signifi- polyacrylamide gel electrophoresis gel, are shown in SI Appendix,Fig.S1. cant conformational change to accommodate the hydrogen Steady-State Activity. The steady-state activity of purified cytochrome aa -600 bonding to 3-iodo-HQNO (and HQNO). A similar change in 3 I was measured by using an oxygen meter (Clark electrode) in the presence of conformation of H94 would also be necessary to hydrogen menaquinone-reducing type II NADH dehydrogenase from Oceanobacillus bond to the semiquinone state of the substrate. iheyensis. About 200 μM NADH was used to keep 10 μM 2,3-dimethyl-1,4- naphtoquinone reduced in the presence of the type II NADH dehydrogenase. Although it is known that the DDM-solubilized cytochrome By adding 0.05 μMcytochromeaa3-600, the reaction was performed under aa3 -600 that is crystallized contains 1 equivalent of menaquinone-7 conditions where the reaction of cytochrome aa3-600 was rate-limiting (22). (22), the weak electron density in the groove defined by TM0, TM1, TM2, and TM3 cannot be definitely assigned as being due to Protein Crystallization. Crystallization was performed in MRC-Maxi 48-well the bound substrate. Assuming that this electron density is due to plates by using the sitting-drop vapor-diffusion method. Two microliters of menaquinone-7, the location is consistent with the oxidized form of protein solution containing 7 mg/mL cytochrome aa3-600 in 20 mM Tris (pH the substrate being trapped bound to the enzyme in the presence of 7.8), 50 mM NaCl, and ∼0.5% DDM was mixed with 2 μL of well solution DDM in a way that neither alters the binding of HQNO nor is containing 0.1 M calcium chloride, 0.1 M Tris (pH 6.3), and 10 to 13% polyethylene glycol monomethyl ether 2000 (PEG 2000 MME) (vol/vol). The perturbed by the binding of HQNO. The simultaneous binding of crystallization drop was incubated at 22 °C against 100 μL of well solution. HQNO and a long-chain quinone species is also observed with long-chain ubiquinone in DDM-solubilized cytochrome bo3 and is an artifact due to the poor solubility of the long-chain quinones in the detergent micelle (26). Under native conditions in a phos- pholipid bilayer, one would not expect the oxidized quinone to remain tightly bound to the enzyme, since it is the product of the reaction catalyzed by the enzyme. In DDM-solubilized cytochrome aa3-600 without the HQNO inhibitor, a 1-electron reduction of the bound (trapped) menaquinone-7 results in the formation of a bound semiquinone stabilized by direct interaction with R70I, D74I, and H94I (21, 22). This could occur easily by a small change of the position of the bound menaquinone within the binding groove, along with the shift in the side chain of H94I (Fig. 3) In summary, the current work clarifies the major structural modifications that have evolved to allow the respiratory heme- c copper oxygen reductases that utilize reduced cytochrome as a Fig. 4. (A) F − F map of the cytochrome aa -600–HQNO complex con- substrate to directly oxidize quinol from the membrane-bound o c 3 toured at 2.5 σ.(B) Fo − Fc map of the cytochrome aa3-600–3-iodo-HQNO pool. The critical structural alterations are 1) the addition of complex. The magenta map representing the position of iodine atom is TM0, which forms part of a cleft that facilitates binding of the contoured at 7.0 σ, and the white map is contoured at 3.0 σ.

4of5 | www.pnas.org/cgi/doi/10.1073/pnas.1915013117 Xu et al. Downloaded by guest on September 28, 2021 The best plate-like crystals of a maximum size of 200 × 80 × 30 μm appeared residues, accounting for 85% of the total residues. Structures of the cy-

after 20 d and reached final size after 30 d. tochrome aa3-600/HQNO complex and the cytochrome aa3-600/iodoHQNO complex were solved by molecular replacement using the native cytochrome

Data Collection and Processing. Crystals were transferred to a cryoprotectant aa3-600 structure as search model, and the ligands were manually fitted by solution comprising 0.1 M calcium chloride, 0.1 M Tris (pH 6.3), and 27% PEG using COOT (32). Structure refinement was performed by using Phenix (31). 2000 MME (vol/vol), then flash frozen in liquid nitrogen. Each of the 3 X-ray Membrane bilayer was generated with CHARMM-GUI Membrane Builder (33), datasets was collected from a single crystal at 100 K at BL17U1 of the and figures of the models were created with PYMOL (34). Shanghai Synchrotron Radiation Facility. Images were processed with Mosflm

(29). The crystals belonged to space group P21, with 2 molecules (complexes) Data Availability. The coordinates for the model of cytochrome aa3-600 from B. per asymmetric unit (SI Appendix, Table S1). subtilis have been deposited in the Protein Data Bank, https://www.rcsb.org (PDB ID codes 6KOB, 6KOC, and 6KOE). Phasing, Model Building, and Refinement. The structure of cytochrome aa3-600 was solved by molecular replacement implemented in Phaser (30). Coordi- ACKNOWLEDGMENTS. We thank Dr. Andrew Leslie for advice on the nates of subunits I and III of cytochrome bo from E. coli (PDB ID code 1FFT) 3 manuscript and the Shanghai Synchrotron Radiation Facility beamline BL17U1 were used as the original search model. Well-defined electron density was for access to their synchrotron facilities. This work was supported by National revealed around the 2 subunits in the search model and fitted by using a Key R & D Plan for Precision Medicine Research Grant 2016YFC0905900; the poly-alanine model from the corresponding model (PDB ID code 1FFT). The Priority Academic Program Development of Jiangsu Higher Education Institu- combined model was used as the starting model for Phenix Autobuild (31). tions (Integration of Chinese and Western Medicine); and Jiangsu Specially The phase was gradually improved, indicated by the better electron density Appointed Professor Funding. Portions of the research were also supported of the Heme farnesyl tail and the side chains of subunits II and IV. Sub- by Chemical Sciences, Geosciences and Biosciences Division, Office of Basic sequently, models were manually built into the well-defined electron den- Energy Sciences, Office of Sciences, US Department of Energy Grant DE- sity by using COOT (32). The final model of 2 complexes contained 2,228 FG02-87ER13716 (to R.B.G.).

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