Figure S1 (separate pdf): Tree topologies used in Table S1
Table S1: Tree statistics comparison using the approximately unbiased (AU) test
Table S2: Purification of NADH dehydrogenase
Table S3: Relative activities of NADH dehydrogenase with different electron acceptors
Table S4: Km analyses
Material and methods for enzyme assays and purification
Figure S1: Tree topologies used in Table S1
Tree 1 is the best tree found using p4 18 and recoded data and places the Trichomonas protein inside the mitochondrial clade (Figure 4 in the manuscript). Tree 5 is the best tree found using MrBayes 3.0B4
29 and non-recoded data and it places the Trichomonas protein outside the mitochondrial clade. Trees 2–4 are trees found during the MrBayes analysis of non-recoded data that place the Trichomonas protein inside the mitochondrial clade and cannot be rejected using the AU test 16 .

Table S1. Comparison of trees showing alternative phylogenetic positions for the Trichomonas vaginalis Tvh-47 protein using the approximately unbiased (AU) test.
A) Tree optimization for amino acid data recoded into 6 categories and analyzed using a dataspecific 6x6 general time reversible (GTR) matrix
Branch lengths, gamma shape parameter, proportion of invariant sites, and GTR matrix were all optimised for their likelihoods on the candidate trees. The likelihood of each tree is expressed as the log likelihood (lnL).
Tree lnL diff lnL P Significant at Trichomonas protein
.
1) -8309.3
2) -8324.0
3) -8322.3
4) -8324.7
5) -8329.2
ML tree
14.7
13.0
15.4
0.898
0.186
0.280
0.127
19.9 0.156
5% (0.05) level?
—
NO
NO
NO inside mitochondrial clade?
YES
YES
YES
YES
NO
__________________
NO
.
<—see Fig. 4
_________ _.
B) Tree optimization for non-recoded data using the empirical WAG matrix
Branch lengths, gamma shape parameter and proportion of invariant sites were all optimised for their likelihoods on the candidate trees.
Tree lnL diff lnL P
1) -18674.9
2) -18644.7
3) -18644.5
4) -18647.5
5) -18630.6
44.3
14.2
13.9
16.9
ML tree
0.025
0.341
0.310
0.334
0.742
Significant at 5% Trichomonas protein
.
(0.05) level? inside mitochondrial clade?
.
YES
NO
NO
NO
—
YES
YES
YES
YES
NO
<— see Fig. 4
.

Table S2 . Purification of NADH dehydrogenase from T. vaginalis hydrogenosomes. The NADH dehydrogenase activity was determined using 100  M 2,6-dichloroindophenol (DCIP) and 3.3 mM
NADH. Details of purification are given in Supplementary Material.
Fraction Total Total Specific
Protein
(mg) activity
(U) activity
(U/mg)
Hydrogenosomes 34.65
Hydrogenosomes extract 11.18
MONO S fraction 0.86
Hydroxyapatite fractions 0.23
347.54
302.13
96.08
34
10.03
27.02
111.72
145.28
Purification Yield fold
1
2.7
11
14.5
(%)
100
87
28
9.8

Table S3.
Relative activities of NADH dehydrogenase with different electron acceptors. Potassium ferricyanide, an efficient artificial low-molecular weight electron acceptor was used as a reference substrate. Details of determinations are given in Supplementary Material.
Electron acceptor Relative activity (%)
Potassium ferricyanide
Coenzyme Q1
Methyl viologen
DCIP
T. vaginalis ferredoxin
Yeast ferredoxin
100
39
38
21
7
1

Table S4.
Michaelis constants of NADH dehydrogenase for NADH and electron acceptors.
K m for potassium ferricyanide, coenzyme Q
1
and Trichomonas vaginalis ferredoxin was determined with 200 µM NADH. K m for NADH was determined with 200 µM potassium ferricyanide, 200 µM coenzyme Q
1
and approx. 60 µM ferredoxin. The numbers are means + standard deviation of at least three independent determinations (number of determinations in parentheses).
Reaction Km donor
[µM] [µM]
Km acceptor
NADH —> Ferricyanide
NADH —> Coenzyme Q1
NADH —> Ferredoxin
21.2 ± 1.5 (4)
50.5 ± 3.2 (3)
49.3 ± 17.4 (7)
293.5 ± 19.7 (3)
251.8 ± 11.0 (4)
46.1 ± 7.8 (4)

Supplementary information on enzyme assays and purification of NADH dehydrogenase
The activity of NADH dehydrogenase was determined spectrophotometrically at 25  C. The activity with T. vaginalis ferredoxin was determined in 50 mM KCl, 100 mM Tris-HCl, pH 8 (assay buffer)
340
NADH = 6220 M -1 cm -1 ).
The specificity of ferredoxin reduction was confirmed by recording the spectra between 260-650 nm upon addition of purified enzyme into the assay mixture under anaerobic conditions. Activity with methyl viologen was determined in the assay buffer with 0.5% mercaptoethanol, 3.3 mM NADH and 1
600
= 6300 M -1 cm -1 ) under anaerobic conditions. Activity with other electron acceptors was determined using 100 µM 2,6-dichloroindophenol (DCIP), 3.3 mM NADH
( 
600
= 21000 M -1 cm -1 ); 980 µM potassium ferricyanide, 270 µM NADH ( 
410
= 1020 M -1 cm -1 ); 250 µM coenzyme Q
1
(dissolved in 96% ethanol), 330 µM NADH ( 
= 6220 M -1 cm -1 ); approx. 65 µM yeast ferredoxin, 200 µM NADH ( 
340
= 6220 M -1 cm
340
-1 ).
One unit of enzyme activity was defined as the amount of protein catalyzing the consumption of one micromole of substrate, or the formation of one micromole of product per minute. The effect of the complex I inhibitor rotenone was examined with purified hydrogenosomes preincubated with 50 µM rotenone for 5 min, in the assay buffer using DCIP as well as coenzyme Q
1
as electron acceptor.
Kinetic parameters were calculated using double-reciprocal plots according to Lineweaver-Burk.
Percoll-purified hydrogenosomes were washed with ST buffer and the pellet was resuspended in 10 mM KH
2
PO
4
-KOH, pH 6.8 (buffer A). Octylglucoside was added to final 2% (w/v) concentration, incubated on ice for 60 min and centrifuged at 240 000 x g for 30 min. NADH dehydrogenase was purified by liquid chromatography using the Biologic HR system (Bio-Rad). The supernatant of octylglucoside-treated hydrogenosomes was loaded onto a MONO S cation-exchange column
(Amersham Biosciences) equilibrated with buffer A, and the enzyme was eluted with a linear gradient
(0 to 1 M) of NaCl in buffer A. Active fractions were pooled and loaded onto a CHT 5-I hydroxyapatite

column (Bio-Rad) equilibrated with buffer A and the enzyme was eluted with a linear gradient (0 to 0.5
M) of 0.5 M KH
2
PO
4
-KOH, pH 6.8.