1

SI Appendix

(Shiba et al.)

SI Materials and Methods

Synthesis of 5-chloro-3-[(2E,6E)-8-hydroxy-3,7-dimethylnona-2,6-dienyl]-2,4-

dihydroxy-6-methyl-benzaldehyde. (E,E)-2,6-Dimethyl-8-(tetrahydropyran-2-

yloxy)octa-2,6-dienal (1) was reacted with MeLi (2 eq) in THF at –85 ºC to give (E,E)-

3,7-dimethyl-9-(tetrahydropyran-2-yloxy)nona-3,7-dien-2-ol. The secondary alcohol

was acetylated with acetic anhydride (2.5 eq) in chloroform in the presence of Et3N (3.0

eq), and DMAP (0.1 eq) at 25 ºC (two steps, 93% yield). The acetate was treated with

pyridinium p-toluenesulfonate (0.4 eq) in methanol at 55 ºC to give (E,E)-8-hydroxy-

1,2,6-trimethylocta-2,6-dienyl acetate (96% yield). The primary alcohol was brominated

with CBr4 (3 eq) and (n-C8H17)3P (3 eq) in ether at 0 ºC. Coupling of the bromide with

3-chloro-4,6-dihydroxy-2-methylbenzaldehede was performed by using a modification

of the previously reported method (2) to produce (E,E)-8-(3-chloro-5-formyl-2,6-

dihydroxy-4-methyl)phenyl-1,2,6-trimethylocta-2,6-dienyl acetate (two steps, 22%

yield). After hydrolysis of acetate with K2CO3 (4.5 eq) in methanol at 25 ºC, the final

product was purified by silica gel column chromatography (hexane/AcOEt, 5:1, 70%

yield). 1H NMR (CDCl3, 400 MHz) δ 1.20 (d, J=6.2 Hz, 3H, CH(OH)CH3), 1.48 (br s,

1H, OH), 1.59 (s, 3H, CH3), 1.78 (s, 3H, CH3), 2.03-2.07 (m, 2H, CH2), 2.08-2.16 (m,

2H, CH2), 2.60 (s, 3H, Ar-CH3), 3.40 (d, J=7.0 Hz, 2H, Ar-CH2), 4.17 (q, J=6.2 Hz, 1H,

CHOH), 5.21 (t, J=7.0 Hz, 1H, CH=C), 5.32 (t, J=6.6 Hz, 1H, CH=C), 6.61 (s, 1H, Ar-

OH), 10.14 (s, 1H, CHO), 12.71 (s, 1H, Ar-OH). IR (KBr) 3200-3500, 1616 cm-1.

Crystallization. The oxidized form of alternative oxidase from Trypanosoma brucei

brucei was expressed, purified and crystallized as previously described (3, 4). We

2

modified the crystallization condition and obtained crystals suitable for X-ray analysis

using the hanging-drop vapor-diffusion method at 20 ºC. A 1 µl droplet of 3-5 mg ml-1

protein solution in 20 mM Tris–HCl pH 7.3, containing 0.8% (w/v) n-octyl β-D-

glucopyranoside (OG), 0.4% (w/v) tetraethylene glycol monooctylether (C8E4), 20 mM

MgSO4, 1 mM ammonium Iron (II) sulfate and 20% (v/v) glycerol was mixed with the

same amount of reservoir solution and equilibrated against 200 µl reservoir solution

(28–34% (w/v) PEG 400, 100 mM imidazole buffer pH 7.4, 500 mM potassium

formate) to give crystals of TAO.

Data collection and phasing. The crystal of TAO soaked briefly in the cryo-protectant

solution (50 % (w/v) PEG 400, 100 mM imidazole buffer pH 7.4, 500 mM potassium

formate, 0.8 % OG, 0.4% (w/v) C8E4) was flash-frozen in liquid nitrogen. For phasing

by SAD method, anomalous scattering effects caused by Fe were measured to 3.2 Å

resolution using an X-ray with wavelength of 1.7390 Å at KEK-PF beamline BL17A

(Tsukuba, Japan). The data set was processed and scaled with HKL2000 (5). SOLVE

program (6) was used to locate and refine four “diiron sites” (FOM=0.195). RESOLVE

(7) was used for solvent flattening assuming 62% solvent content (FOM=0.645). The

map was clear to trace the TAO molecules. Initial model was built using RESOLVE (7)

and BUCANER (8). Detailed analysis of the diffraction data shows that the crystal of

Fe-SAD data was pseudo-hemihedral twinning and belong to space group C2 with unit

cell parameters a = 148.4, b = 223.0, c = 62.7 Å and β = 114.9°, and four monomers in

the asymmetric unit. Amplitude-based twin-refinement using REFMAC5 (9) decreased

Rwork/Rfree drastically from 0.307/0.363 to 0.250/0.310. X-ray diffraction data of ligand-

free TAO crystal was collected to 2.85 Å resolution at 100 K at SPring-8 beamline

BL41XU (Harima, Japan). The crystals of the TAO-inhibitor complex were prepared by

3

soaking TAO crystals in the cryo-protectant solution supplemented with 1 mM

AF2779OH for 150 min and 1 mM CCB for 90 min at 20 ºC. The data were collected to

2.6 and 2.3 Å resolution for TAO-AF2779OH and TAO-CCB, respectively at 100 K at

BL44XU (SPring-8). All data sets were processed and scaled with HKL2000 (5).

Refinement. The initial model of inhibitor-free TAO was determined by molecular

replacement (MR) using the model obtained by SAD (3.2 Å resolution) as a search

model. The program, Phaser (10) in CCP4i was used for MR. The models of the TAO-

AF derivative complexes were successively obtained by MR using the refined structure

of TAO. The models of ligand-free TAO and TAO-AF2779OH complex were re-built

with reference to the well refined model of TAO-CCB complex at 2.3 Å resolution.

Manual re-building and crystallographic refinement of all structures were performed

using the COOT (11) and REFMAC5 (9). All structures were refined by amplitude-

based twin-refinement in REFMAC5 (9) to final Rwork/Rfree values of 0.192/0.247 (twin

fraction of 0.476), 0.214/0.256 (twin fraction of 0.552) and 0.185/0.227 (twin fraction

of 0.527) for ligand-free TAO, TAO-AF2779OH, TAO-CCB, respectively.

Stereochemistry of the refined structures, validated by PROCHECK (12) and

Ramachandran plots indicated that no amino acid residues were detected in

energetically unfavorable regions. Out of 329 amino acid residues constituting the

whole TAO protein, chains A, B, C and D in the asymmetric unit are comprised of

residues: 31-297, 32-299, 32-297 and 32-297, respectively for the ligand-free; 30-295,

32-296, 31-296 and 30-295, respectively for the TAO-AF2779OH complex; and 32-295,

31-296, 32-297 and 32-294, respectively for TAO-CCB complex. Data collection and

structural refinement statistics are summarized in Table S1. Figures showing protein

structures were prepared with the graphics program PyMol (http://www.pymol.org/).

90º

A

B

C

90º

90º

BA C

D

D'C'

BA CDD'

C'

AB C

DD'C'

A B CDD'

C'

ACD A' D' A'

Fig. S1. Crystal packing of TAO molecule. Crystal packing of chains A, B, C and D in the asymmetric

units of (A) AF2779OH-free TAO, (B) TAO-AF2779OH and (C) TAO-CCB complex viewed from two

directions perpendicular to each other. Chains A, B, C and D are colored in light green, light pink, cyan and

yellow. Diiron and hydroxo atoms are shown as the magenta spheres and the bound AF2779OH or CCB as

red stick. In (A) and (B), chains C' and D' are related to chains C and D, respectively, by crystallographic 2-

fold axes. A-B, C-C' and D-D' pairs form dimers in A, whereas A-B, C-D and C'-D' pairs in B. Chains C' and

D' are colored in dark blue and orange, respectively. In (C), chains A' and D' are related to chains A and D,

respectively, by crystallographic 2-fold axes. A-A', D-D' and B-C pairs form dimers in C.

BAC

DD'

A'

B ACD

D' A'

4

αααα1

αααα2

αααα3

αααα4αααα5 αααα6

C

N

S3

S4

S1

αααα1

αααα2

αααα3

αααα4

αααα5

αααα6

C N

S3

S4S1

N-terminal armN-terminal arm

50 Å

30 Å 30 Å

35 Å90º

S2

S2

Fig. S2. Monomer structure of TAO. Ribbon representation of chain A viewed as in Fig. 1A. Monomer

structure of TAO viewed roughly perpendicular (left) and parallel (right) to the helix axes. Chain A is

colored in rainbow from blue (N-terminus) to red (C-terminus). Except for the N-terminal arm, each

monomer is shaped as a compact cylinder (50×30×35 Å). The diiron (Fe-OH--Fe) is drawn as magenta

spheres.

5

Q187

H138

R163

M131

M135

L166I183

A159

M167R143

H138

M131

M135

L142

L139

R147M145

S141D148

A159L156

R180

L156

R143

H138

L142L139

R147M145

S141

D148Q187

R163

L166I183

M167

Fig. S3. Dimer interface of the TAO dimer. The upper panel represents the TAO dimer formed by the A-B

pair of the AF2779OH-free TAO viewed from the same direction as Fig. 1A (left panel), and the lower is the

‘open book’ representation of the upper. Chains A and B are colored in light green and light pink,

respectively. Residues conserved in all eight and more than four species (Fig. S5) are colored in red and

orange, respectively.

R180

6

90º 90º 90º

Fig. S4. The proposed membrane binding model of the TAO dimer viewed from four directions. The

R106R180 R203* R106R180* R203 R106R106*R180* R180 R106*R106*

Fig. S4. The proposed membrane binding model of the TAO dimer viewed from four directions. The

colour codes are as in Fig. 1C. Conserved basic residues located on the interface between TAO and the

membrane are colored in blue. Residues name are labeled black (asterisks denotes in chain B).

7

αααα6 S4

S1

αααα1 αααα2S2

S3α4α4α4α4 α5α5α5α5αααα3 S3

Fig. S5. Multiple alignment of amino acid sequences of alternative oxidases. The alignment was

produced from amino acid sequences from Trypanosoma brucei brucei (protozoa), Cryptosporidium parvum

(protozoa), Sauromatum guttatum (plant), Chlamydomonas reinhardtii (alga), �ovosphingobium

aromaticivorans (α-proteobacteria), �eurospora crassa (mold), Candida albicans (anascosporogenous

yeast) and Ciona intestinalis (chordata). The secondary structure elements identified in the TAO structure

are also shown. Helices α1 and α4, which form the hydrophobic region on the molecular surface of TAO

and are proposed to be concerned in membrane binding, are colored in yellow. Helices α2, α3, α5 and α6,

which form the four helix bundle to accommodate the diiron centre, are colored in cyan. Amino acid

residues coordinating the diiron centre and interacting with AF2779OH are indicated by * and +, respectively.

Cyan and pink colours stand for conservation of AOX residues in all 8, and over 4 organisms, respectively.

8

C D

A B

E213

E266

Fe1Fe2

OH-

E123

E162

H165H269

E213

E266

Fe1Fe2

OH-

E123

E162

H165H269

E213Fe1

Fe2

OH-

E123

E162

E213 Fe1Fe2

OH-E123

E162

E266H165H269

E266 H165H269

Fig. S6. The structure of diiron sites. Interaction between the diiron molecule and TAO with sigma-A

weighted electron density map (Fo-Fc) calculated from the refined model of ligand-free TAO with the diiron

molecule omitted from the phase calculation. The diiron molecules bound to (A) chain A, (B) chain B, (C)

chain C and (D) chain D are shown as yellow sticks with nitrogen and oxygen atoms colored in blue and red.

The diiron (Fe-OH--Fe) is drawn as magenta spheres. Contour levels are 1.0 σ (blue) and 3.0 σ (orange).

9

Tyr198

Tyr246

Tyr220

His206

Asn161

Fe2OH-

Fe1

AF2779OH

αααα5

S3

αααα3

αααα4

Fig. S7. Conserved tyrosine residues. The bound AF2779OH molecule is shown as red stick. Tyr220

(blue) is located near the diiron and AF2779OH, but Tyr198 and Tyr246 (orange) are far from both the diiron

(magenta spheres) and AF2779OH. The interacting residues of these tyrosines are shown as light pink stick

models and dash lines stand for hydrogen bonds.

10

D

A B

R118T219

F99

E215

C95

R96

L212

E213

L122

A216

Y220

E266

Fe1

Fe2 OH-E123

E162

F121

H165H269

R118

T219

F99

E215

C95 R96

L212

E213

L122

A216

Y220

E266

Fe1Fe2

OH-

E123

E162

C119

H165H269

M190V125

R118

T219

F99

E215

R96

L212

C119

F121

R118

T219

E215

C95R96

L122V92

C95

C

C119

L212

E213

L122

A216Y220

E266

Fe1Fe2

OH-E123

E162

H165H269

M190

F121

L212

E213

A216

Y220

E266

Fe1Fe2

OH-

E123

E162

C119

H165H269

V125

Fig. S8. Interaction between TAO and the bound AF2779OH molecules and their electron density

maps. Interaction between the bound AF2779OH molecule and TAO with sigma-A weighted electron

density map (Fo-Fc) calculated from the refined model of TAO-AF2779OH complex with the AF2779OH

molecule omitted from the phase calculation. AF2779OH molecules bound to (A) chain A, (B) chain B, (C)

chain C and (D) chain D are shown as pink sticks with nitrogen, oxygen and chlorine atoms colored in blue,

red and green, respectively. The diiron (Fe-OH--Fe) is drawn as magenta spheres. Contour levels are 1.0 σ

(blue) and 3.0 σ (orange). 11

R118T219

F99

E215

C95 R96

L212

E213

L122A216

C119

E266

Fe1Fe2

OH-

E123

E162

H165H269

V92

M190

R118T219

F99

E215

R96

L212

E213

L122

A216C119

E266

Fe1Fe2

OH-

E123

E162

H165

H269

M190

F121

R118T219

F99

C95

R96

L122

R118T219

F99C95 R96

L122C119V92

BA

C95

C D

E215E215

L212

E213

A216C119

E266

Fe1Fe2

OH-

E123

E162

H165H269

V125Y220 L212

E213

A216

E266

Fe1Fe2

OH-E123

E162

H165H269

V92

V125

Y220

Fig. S9. Interaction between TAO and the bound CCB molecules and their electron density maps.

Interaction between the bound CCB molecule and TAO with sigma-A weighted electron density map (Fo-

Fc) calculated from the refined model of TAO-CCB complex with the CCB molecule omitted from the

phase calculation. CCB molecules bound to (A) chain A, (B) chain B, (C) chain C and (D) chain D are

shown as pink sticks with nitrogen, oxygen and chlorine atoms colored in blue, red and green, respectively.

The diiron (Fe-OH--Fe) is drawn as magenta spheres. Contour levels are 1.0 σ (blue) and 3.0 σ (orange).

L212

12

R118

T219

F99

E215

C95

R96

L212

E213

L122

A216

C119

Fe1Fe2

OH-

E123

M190

F121

V125

Y220

V92

R118

T219

F99

E215

C95

R96

L122

C119E123

M190

F121

V125V92

B

A

R118

T219

F99

E215

C95

R96

L212

E213

L122

A216

C119

Fe1Fe2

OH-

E123

M190

F121

V125

Y220

V92

R118

T219

F99

E215

C95

R96

L122

C119E123

M190

F121

V125V92

L212

E213

A216

Fe1Fe2

OH-

E123V125

Y220

Fig. S10. Stereo view of superposed inhibitor binding site of all chains of the complex structures. (A)

AF2779OH-TAO and (B) CCB-TAO. Chains A, B, C and D are colored in light-green, light-pink, cyan and

yellow, respectively.

L212

E213

A216

Fe1Fe2

OH-

E123V125

Y220

13

AF2779OH IC50= 0.48 nM

CCB IC50 = 0.20 nM

K2-9 IC50 = 250 nM

K4-9 IC50 = 4000 nM

Fig. S11. IC50 of the AF derivatives. IC50s of the AF derivatives with (AF2779OH and CCB) and without

(K2-9 and K4-9) the aldehyde group.

14

Fig. S12. Stereo view of inhibitor-free TAO and AF2779OH-TAO complex around active site. Inhibitor-

Y220

E213

E162

E123E266

H269H165

Fe2

Fe1

OH-A216

L212L122

E215

C119

C95

R96

F99

R118T219

F121 Y220

E213

E162

E123E266

H269H165

Fe2

Fe1

OH-A216

L212L122

E215

C119

C95

R96

F99

R118T219

F121

Fe1 Fe1OH- OH-

Fig. S12. Stereo view of inhibitor-free TAO and AF2779OH-TAO complex around active site. Inhibitor-

free TAO and AF-2779OH-TAO complex are shown in light-pink and light-green sticks, respectively. Unlike

amino acid residues, the diirons poorly superimposed on each other.

15

2.4 Å

3.3 Å

Fe1

2.5 Å

W147

D265

H165

Y220H269

E162

E213E123

E266

T219A216

E215

R96

V92

C95

F99F121

V125

M190

R118

L122L212

C119

Fig. S13. Putative ubiquinol binding channels in CCB-TAO complex. Two hydrophobic cavities found

by CAVER protein-analysis software (13) are shown by green and orange. Proposed residues involved in

electron transfer are shown as orange sticks.

16

R203

Y211

Y220

E229

R203

Y220

E229

Y211

R203

Y220

E229

Y211

BA C

Inhibitor-free AF2779OH-TAO

Fig. S14. Omit electron density maps around helix5 of (A) ligand-free , (B) AF2779OH -TAO complex

and (C) CCB-TAO complex. Contour level is 1.0σ (blue).

CCB-TAO

E229E229

E229

17

18

Table S1. Data collection and refinement statistics

Fe-peak inhibitor-free TAO TAO-AF2779OH TAO-CCB

Data collection

Space group C2 C2 C2 C2

Cell parameters

a / b / c (Å) 148.4 / 223.0 / 62.7 261.3 / 63.1 / 136.5 152.3 / 219.7 / 63.5 228.0 / 137.9 / 63.1

α / β / γ (°) 90 / 114.9 / 90 90 / 121.4/ 90 90 / 114.9 /90 90 / 106.1 / 90

Wavelength (Å) 1.7390 1.0000 0.9000 0.9000

Resolution (Å) 50.0 – 3.2 (3.26 – 3.2) 50.0 – 2.85 (2.9 – 2.85) 50 – 2.6 (2.64 – 2.6) 50 – 2.3 (2.34-2.3)

Rmerge I 0.136 (0.604) 0.084 (0.515) 0.124 (0.614) 0.095 (0.506)

I/σ(I) 6.8 (1.1) 9.6 (1.5) 8.6 (1.3) 7.7 (2.6)

Completeness (%) 91.1 (43.0) 89.3 (54.2) 93.4 (85.3) 99.0 (99.6)

Redundancy 4.7 (3.7) 3.4 (2.8) 2.7 (2.1) 3.3 (3.2)

Refinement

Resolution range (Å) 30.0 – 2.85 30 – 2.6 30 – 2.3

Number of reflections 37,533 51,307 77,977

Twin ratios 0.476 0.552 0.527

Rwork/Rfree 0.192 / 0.247 0.214 / 0.256 0.185 / 0.227

No. atoms

Protein 8,675 8,645 8616

diiron 12 12 12

inhibitor - 96 88

solvent 31 131 292

B-factor (Å2)

Protein 84.3 42.9 42.7

diiron 64.4 29.3 29.5

inhibitor - 47.4 45.9

solvent 65.0 36.0 40.4

R.m.s. deviation

Bond length (Å) 0.010 0.008 0.009

Bond angle （°） 1.56 1.32 1.41

Values in parentheses are for the highest resolution shell.

19

OO

Fe1Fe2OH-O

O O O

OO

N

N

His165N

N

His269

Glu123

Glu162

Glu266

Glu213

HO

Tyr220Oεεεε2 Oεεεε1

Oεεεε2 Oεεεε1

Oεεεε1 Oεεεε1

Oεεεε2 Oεεεε2

Oηηηη

Nδδδδ1 Nδδδδ1

OO

Fe1Fe2OH-O

O O O

OO

N

N

His165N

N

His269

Glu123

Glu162

Glu266

Glu213

HO

Tyr220Oεεεε2 Oεεεε1

Oεεεε2 Oεεεε1

Oεεεε1 Oεεεε1

Oεεεε2 Oεεεε2

Oηηηη

Nδδδδ1 Nδδδδ1

Table S2. Diiron structure

Inhibitor-free TAO

distance (Å) average A B C D (standard deviation) Fe1-Fe2 3.50 2.99 3.42 3.07 3.25 (22) Glu123Oε1-Fe1 2.04 1.85 2.45 2.17 2.13 (22) Glu123Oε2-Fe1 2.20 2.10 1.86 2.01 2.04 (12) Glu162Oε1-Fe1 2.52 1.85 2.48 2.12 2.24 (27) Glu266Oε1-Fe1 1.96 2.21 1.80 1.80 1.94 (17) Glu213Oε1-Fe2 2.51 1.80 2.34 1.98 2.16 (28) Glu213Oε2-Fe2 2.30 2.16 1.95 2.23 2.16 (13) Glu162Oε2-Fe2 2.34 1.82 1.91 2.31 2.10 (23) Glu266Oε2-Fe2 2.14 2.49 2.13 2.20 2.24 (15) His165Nδ1-Fe1 3.28 3.44 4.00 3.32 3.51 (29) His269Nδ1-Fe2 4.40 3.83 4.32 3.83 4.10 (27) Tyr220Oη-Fe1 4.01 4.00 3.32 3.85 3.80 (28) TAO-AF2779OH complex

distance (Å) average A B C D (standard deviation) Fe1-Fe2 3.75 3.11 3.48 3.70 3.51 (25) Glu123Oε1-Fe1 2.10 2.36 2.31 2.15 2.23 (11) Glu123Oε2-Fe1 2.47 2.15 2.35 2.26 2.31 (12) Glu162Oε1-Fe1 2.03 1.96 2.01 2.41 2.10 (18) Glu266Oε1-Fe1 2.08 1.87 2.19 1.89 2.01 (13) Glu213Oε1-Fe2 2.38 2.29 2.13 2.64 2.36(18) Glu213Oε2-Fe2 2.45 2.21 2.25 2.22 2.28 (10) Glu162Oε2-Fe2 2.07 2.26 2.04 2.36 2.18 (13) Glu266Oε2-Fe2 2.20 1.92 2.21 2.06 2.10 (12) His165Nδ1-Fe1 2.21 2.50 2.32 2.31 2.34 (10) His269Nδ1-Fe2 4.40 4.25 4.09 4.55 4.32 (17) Tyr220Oη-Fe1 4.86 4.73 4.59 4.56 4.69 (12)

20

TAO-CCB complex

distance (Å) average A B C D (standard deviation) Fe1-Fe2 3.20 3.54 3.28 3.54 3.39 (15) Glu123Oε1-Fe1 2.26 2.20 2.25 2.14 2.21 (5) Glu123Oε2-Fe1 2.16 2.17 2.31 2.13 2.19 (7) Glu162Oε1-Fe1 2.02 2.88 1.99 1.96 2.21 (39) Glu266Oε1-Fe1 2.09 1.84 2.34 1.91 2.05 (19) Glu213Oε1-Fe2 2.20 2.16 2.11 2.28 2.19 (6) Glu213Oε2-Fe2 2.11 2.27 2.37 2.12 2.22 (11) Glu162Oε2-Fe2 1.98 2.14 2.07 1.87 2.02 (10) Glu266Oε2-Fe2 2.17 2.00 2.18 2.26 2.15 (9) His165Nδ1-Fe1 2.38 2.33 2.57 2.42 2.43 (9) His269Nδ1-Fe2 4.14 4.53 3.93 4.19 4.20 (22) Tyr220Oη-Fe1 4.72 4.67 4.61 4.66 4.67 (4)

Table S3. Interaction between TAO and

AF2779OH residuesChain A aromatic head C2-OH Arg118 Arg118 Thr219 Oring Arg118

Cys119Leu122

Glu123Leu212

Ala216 Thr219

Tyr220isoprenoid tail Cys95

Arg96 Phe99 Phe121 Leu122

Leu212Glu215

Chain B aromatic head C1=O Cys119SC2-OH Arg118 Thr219 Oring Arg118

Cys119Leu122

Glu123Leu212

Ala216 Thr219

Tyr220isoprenoid tail Cys95

Arg96 Phe99 Leu122 Val125

Met190Glu215

between TAO and AF2779OH

residues within 4 Å distance (Å) interaction

118Nη2 3.0 hydrogen bond118Nε 3.1 hydrogen bond

Thr219 Oγ1 2.6 hydrogen Arg118 van der WaalsCys119 van der Waals

122 van der WaalsGlu123 van der Waals

212 van der Waals216 van der Waals

Thr219 van der 220 van der Waals

Cys95 van der WaalsArg96 van der WaalsPhe99 van der WaalsPhe121 van der WaalsLeu122 van der Waals

212 van der WaalsGlu215 van der Waals

Cys119Sγ 3.2 hydrogen bond118Nε 3.0 hydrogen bond

Thr219 Oγ1 2.9 hydrogen bondArg118 van der WaalsCys119 van der

122 van der Glu123 van der Waals

212 van der Waals216 van der Waals

Thr219 van der Waals 220 van der Waals

Cys95 van der Waals Arg96 van der Waals Phe99 van der Waals Leu122 van der Waals Val125 van der Waals Met190 van der Waals Glu215 van der Waals

21

nteraction

hydrogen bond hydrogen bond hydrogen bond van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact

hydrogen bond hydrogen bond hydrogen bond van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact

22

Table S3. Interaction between TAO and AF2779OH (continued) AF2779OH residues within 4 Å distance (Å) interaction Chain C aromatic head C2-OH Arg118Nη2 2.6 hydrogen bond

Arg118Nε 3.2 hydrogen bond Thr219 Oγ1 2.5 hydrogen bond C4-OH Leu212O 2.6 hydrogen bond ring Arg118 van der Waals contact

Cys119 van der Waals contact Leu122 van der Waals contact Leu212 van der Waals contact Glu213 van der Waals contact Glu215 van der Waals contact

Ala216 van der Waals contact Thr219 van der Waals contact

Tyr220 van der Waals contact isoprenoid tail Cys95 van der Waals contact

Arg96 van der Waals contact Phe99 van der Waals contact Phe121 van der Waals contact Leu122 van der Waals contact

Met190 van der Waals contact Glu215 van der Waals contact

Chain D aromatic head C1=O Cys119Sγ 3.3 hydrogen bond C2-OH Arg118Nη2 2.8 hydrogen bond

Arg118Nε 2.9 hydrogen bond Thr219 Oγ1 2.8 hydrogen bond ring Arg118 van der Waals contact

Cys119 van der Waals contact Leu122 van der Waals contact

Glu123 van der Waals contact Leu212 van der Waals contact

Ala216 van der Waals contact Thr219 van der Waals contact

Tyr220 van der Waals contact isoprenoid tail Val92 van der Waals contact

Cys95 van der Waals contact Arg96 van der Waals contact

Leu122 van der Waals contact Val125 van der Waals contact

Leu212 van der Waals contact Glu215 van der Waals contact

Table S3. Interaction between TAO and

CCB residuesChain A aromatic head C1=O Arg118C2-OH Arg118 Arg118 Thr219 Oring Arg118

Cys119Leu122

Glu123Met190Leu212

Glu213Ala216

Thr219isoprenoid tail Val92

Cys95Arg96

Phe99 Leu122

Leu212Glu215

Chain B aromatic head C2-OH Arg118 Arg118 Thr219 OC4-OH Leu212Oring Arg118

Cys119Leu122

Glu123Leu212

Ala216 Thr219 Glu266

isoprenoid tail Cys95Arg96

Phe99Phe121Leu122Met190Leu212Glu215

between TAO and CCB

residues within 4 Å distance (Å) interaction

Arg118Nε 3.2 hydrogen bondArg118Nη2 2.9 hydrogen bondArg118Nε 2.8 hydrogen bondThr219 Oγ1 2.7 hydrogen bondArg118 van der WaalsCys119 van der

122 van der Waals Glu123 van der Met190 van der Waals

212 van der Waals Glu213 van der

216 van der Waals Thr219 van der Val92 van der Waals Cys95 van der Waals Arg96 van der Waals Phe99 van der Waals Leu122 van der Waals

212 van der Waals Glu215 van der Waals

Arg118Nε 3.1 hydrogen bondArg118Nη2 2.8 hydrogen bondThr219 Oγ1 2.6 hydrogen bondLeu212O 3.2 hydrogen bondArg118 van der WaalsCys119 van der

122 van der WaalsGlu123 van der Waals contact

212 van der 216 van der Waals contact

Thr219 van der Waals contactGlu266 van der WaalsCys95 van der Arg96 van der Waals contactPhe99 van der WaalsPhe121 van der WaalsLeu122 van der Waals Met190 van der Waals

212 van der Waals Glu215 van der Waals

23

nteraction

hydrogen bond hydrogen bond hydrogen bond hydrogen bond van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact

hydrogen bond hydrogen bond hydrogen bond hydrogen bond van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact van der Waals contact

24

Table S3. Interaction between TAO and CCB (continued) CCB residues within 4 Å distance (Å) interaction Chain C aromatic head C1=O Cys119Sγ 3.1 hydrogen bond C2-OH Arg118Nη2 2.4 hydrogen bond Thr219 Oγ1 2.4 hydrogen bond ring Arg118 van der Waals contact

Cys119 van der Waals contact Leu122 van der Waals contact

Glu123 van der Waals contact Leu212 van der Waals contact Glu213 van der Waals contact

Ala216 van der Waals contact Thr219 van der Waals contact

Tyr220 van der Waals contact isoprenoid tail Cys95 van der Waals contact

Arg96 van der Waals contact Phe99 van der Waals contact Leu122 van der Waals contact Val125 van der Waals contact

Leu212 van der Waals contact Glu215 van der Waals contact

Chain D aromatic head C1=O Cys119Sγ 3.0 hydrogen bond C2-OH Arg118Nη2 2.6 hydrogen bond Thr219 Oγ1 2.5 hydrogen bond ring Arg118 van der Waals contact

Cys119 van der Waals contact Leu122 van der Waals contact

Glu123 van der Waals contact Leu212 van der Waals contact

Ala216 van der Waals contact Thr219 van der Waals contact

Tyr220 van der Waals contact isoprenoid tail Val92 van der Waals contact

Cys95 van der Waals contact Arg96 van der Waals contact

Phe99 van der Waals contact Leu122 van der Waals contact Val125 van der Waals contact

Glu215 van der Waals contact

25

Table S4. Ubiquinol oxidase activity of TAO mutants overexpressed in E. coli/DhemA strains

Strain Specific activity (%) Function

WT 100 -

R118A 0.2 inhibitor binding

R118Q 0.3 inhibitor binding

L122A 0.2 inhibitor binding

L122N 0.3 inhibitor binding

Y198A* 56 stabilizing the structure, hydrogen bond with His206

E213A* 0 diiron binding

E215A 2.5 inhibitor binding

A216L 0.2 inhibitor binding

A216N 0.2 inhibitor binding

T219V 0.5 inhibitor binding

Y220F 0.5 inhibitor binding, tyrosine radical

Y246A* 5.0 stabilizing the structure, diiron hydrogen network

pET15b 0.1 -

*results from ref. 14.

26

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