THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 43, pp.
32724 –32727, October 27, 2006
Bovine Complex I Is a Complex of 45 Different Subunits*
Received for publication, July 27, 2006 Published, JBC Papers in Press, September 1, 2006, DOI 10.1074/jbc.M607135200
Joe Carroll, Ian M. Fearnley, J. Mark Skehel1, Richard J. Shannon, Judy Hirst, and John E. Walker2
From the Dunn Human Nutrition Unit, The Medical Research Council, Hills Road, Cambridge CB2 2XY, United Kingdom
Mammalian mitochondrial complex I is a multisubunit mem-
brane-bound assembly with a molecular mass approaching 1
MDa. By comprehensive analyses of the bovine complex and its
constituent subcomplexes, 45 different subunits have been
characterized previously. The presence of a 46th subunit was
suspected from the consistent detection of a molecular mass of
10,566 by electrospray ionization mass spectrometry of subunits
fractionated by reverse-phase high pressure liquid chromatog-
raphy. The component was found associated with both the
intact complex and subcomplex I␤, which represents most of
the membrane arm of the complex, and it could not be resolved
chromatographically from subunit SGDH (the subunit of bovine
complex I with the N-terminal sequence Ser-Gly-Asp-His). It
has now been characterized by tandem mass spectrometry of
intact protein ions and shown to be a C-terminal fragment of
subunit SGDH arising from a specific peptide bond cleavage
between Ile-55 and Pro-56 during the electrospray ionization
process. Thus, the subunit composition of bovine complex I has
been established. It is a complex of 45 different proteins plus
non-covalently bound FMN and eight iron-sulfur clusters.
Complex I (NADH ubiquinone oxidoreductase) catalyzes
the first stages of oxidative phosphorylation in mitochondria by
oxidizing NADH in the matrix and transferring the electrons
via flavin mononucleotide and a series of iron-sulfur centers to
ubiquinone in the inner membrane (1, 2). The passage of two
electrons, one at a time, from NADH to ubiquinone is coupled
to the translocation of four protons from the mitochondrial
matrix to the intermembrane space (2, 3). The bovine enzyme is
a membrane-bound assembly of ⬃45 polypeptides with a com-
bined molecular mass approaching 1 MDa together with non-
covalently bound FMN and eight iron-sulfur clusters (4). It has
an L shape with one arm embedded in the membrane and
another, the peripheral arm, orthogonal to it protruding into
the mitochondrial matrix (5, 6). The complex can be dissoci-
ated under mild conditions into subcomplexes. Subcomplex
1␭, which corresponds to the peripheral arm, contains 15 sub-
units and all the known redox cofactors and the NADH binding
site. Subcomplex I␣ is subcomplex I␭ plus nine additional sub-
units (4, 7–11). Subcomplexes I␤ (13 subunits) and I␥ (6 sub-
units) provide most of the rest of the membrane arm (4, 8 –12).
On the basis of the presence of motifs for binding redox centers
* The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 4
Current address: GlaxoSmithKline, Stevenage, Hertfordshire SG1 2NY,
United Kingdom.
2
To whom correspondence should be addressed. Tel.: 44-1223252701; Fax:
44-1223252705; E-mail: walker@mrc-dunn.cam.ac.uk.
This is an open access article under the CC BY license.
32724 JOURNAL OF BIOLOGICAL CHEMISTRY
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
in various subunits and from the conservation of seven subunits
of the complex in mitochondrial DNA sequences, 14 of the
subunits were defined as providing the catalytic “core” of the
enzyme, and the remaining subunits were defined as being
“supernumerary” (2). Subsequently, it has been found that the
simpler bacterial enzymes are composed of homologues of the
core subunits and that the supernumerary subunits have no
bacterial counterparts (13–15). Some of them appear to have
functions that are unrelated directly to the electron transfer and
proton pumping activities of the enzyme, but most of them
have no known function (4, 9, 10, 16 –19). The bovine enzyme
has also provided a model for characterizing the much less
readily available human enzyme (20). Mutations in subunits of
human complex I have been associated with neurological and
neuromuscular diseases (21).
In previous work, the protein subunit compositions of bovine
complex I and subcomplexes I␭, I␣, I␤, and I␥ (2, 4, 8 –12) have
been investigated exhaustively by separating their subunits by a
variety of different methods. They include one-dimensional gel
electrophoresis, two-dimensional gel analysis involving equi-
librium or non-equilibrium isoelectric focusing combined with
SDS-PAGE, reverse-phase HPLC (11), and extraction of hydro-
phobic subunits with organic solvents coupled with hydrophilic
interaction chromatography.3 Thereafter, the purified subunits
were analyzed by Edman degradation and by mass spectromet-
ric methods for measurements of intact protein masses by pep-
tide mass fingerprinting and by amino acid sequencing of pep-
tides by tandem MS4 (9, 10, 22–26). By these means, 45
different polypeptides have been identified in the complex and
allocated to its peripheral and membrane arms. Stable post-
translational modifications of all subunits except ND6 have
been defined also (10, 11, 24, 26 –28).
In numerous experiments, a component with a molecular
mass of 10,566 Da has been detected by ESI-MS analysis of
fractions resulting from reverse-phase HPLC separation of
subunits from intact complex I and from subcomplex I␤.
This component co-eluted consistently with subunit SGDH,
a known subunit of subcomplex I␤ (4, 11, 24). This mass was
assumed to arise from an uncharacterized subunit of com-
plex I (11, 25, 26), and until now, it has resisted character-
ization. No novel sequences were found in digests of appro-
priate fractions with trypsin, chymotrypsin, Asp-N, and
cyanogen bromide or in combined digests with trypsin and
cyanogen bromide (11), and the only components that were
3
J. Carroll, I. M. Fearnley, and J. E. Walker, unpublished work.
The abbreviations used are: MS, mass spectrometry; ESI-MS, electrospray ioni-
zation mass spectrometry; HPLC, high pressure liquid chromatography;
SGDH, the subunit of bovine complex I with the N-terminal sequence
Ser-Gly-Asp-His.
VOLUME 281 • NUMBER 43 • OCTOBER 27, 2006
 Subunit Composition of Bovine Mitochondrial Complex I
found were those arising from subunit SGDH. The 10,566 spectrometer (VG Instruments; Altrincham, UK) was also
mass does not correspond to an N-terminal fragment of sub- used with a cone voltage of 65 V and a capillary voltage of
unit SGDH. These results indicated the presence of an ⬃3000 V. The voltages applied to sample cones or orifices
unknown protein with a modified N terminus and an unusual were chosen for the optimal ionization and transmission of
amino acid composition. Recently, the nature of the unat- myoglobin ions. Alternatively, both MS and tandem MS
tributed mass has been investigated by analysis of multiply experiments were carried out with a hybrid quadrupole
charged protein ions by tandem MS in a quadrupole time- time-of-flight mass spectrometer (Q-TOF1) equipped with a
of-flight mass spectrometer. Partial amino acid sequences Z-spray ion source and a nanoflow electrospray interface
derived from these experiments have demonstrated that the (Waters-Micromass; Manchester, UK) using a spray capil-
10,566 Da component is a fragment of subunit SGDH that lary of 20 ␮M inner diameter, a capillary voltage of 2500 –
arises by specific cleavage of a peptide bond during electro- 2800 V, and sample and extraction cone voltages of between
spray ionization of the protein. 50 and 60 and 4 V, respectively. Argon was used for collision-
induced dissociation.
EXPERIMENTAL PROCEDURES The Sciex API III⫹ triple quadrupole mass spectrometer was
Purification and Analysis of Complex I and Subcomplex I␤ calibrated for measurements of protein mass as described pre-
from Bovine Heart Mitochondria—Complex I was purified viously (9), and the Q-TOF instrument was calibrated with
from extracts of mitochondrial membranes made in the pres- horse heart myoglobin and bovine trypsinogen. Tandem mass
ence of n-dodecyl-␤-D-maltoside (Anatrace; Maumee, OH) (9, spectra were interpreted manually. Leucine and isoleucine res-
11, 12), and subcomplex I␤ was prepared from it as described idues were not distinguished in any of these experiments, and
previously. they were assigned according to known sequences.
The subunits of complex I and subcomplex I␤ were frac-
tionated by reverse-phase HPLC, as described previously RESULTS AND DISCUSSION
(11). Samples were introduced into mass spectrometers by Mass Spectral Analysis of the 10,566-Da Component—The
flow injection (9, 25) or from a nanoelectrospray needle (29). ESI mass spectrum of the fraction from subcomplex I␤ that
Molecular masses of subunits were measured by positive ion gave rise to the 10,566-Da component contained two overlap-
ESI-MS. Mass measurements were made with a PE Sciex API ping series of molecular ions corresponding to the 10,566-Da
III⫹ triple quadrupole mass spectrometer (MDS Sciex; Con- component and to subunit SGDH (11, 24). Ions from subunit
corde, Ontario, Canada) using an orifice potential of SGDH were more intense than those from the 10,566-Da com-
between 70 – 80 V and potentials of ⬃4000 and 650 V on the ponent (Fig. 1). Their relative proportions differed in some
capillary needle and interface plate, respectively, or 600 –700 spectra, but ions from subunit SGDH were usually predomi-
V in nanospray experiments (29). A Bio-Q quadrupole mass nant. The spectrum also contained minor ions that were not
part of either series and are dis-
cussed later.
The multiply charged protein
molecular ions produced by electro-
spray ionization were analyzed
directly by tandem MS. A molecular
ion (1057.5 m/z) containing ten pro-
tons in the ESI-MS spectrum of the
10,566 component was fragmented
by collision-induced dissociation in
a quadrupole time-of-flight mass
spectrometer at low collision
energy, similar to the conditions
used for fragmenting peptide ions.
The resultant fragment ion spec-
trum (Fig. 2) contained several sin-
gly charged ions from m/z 100 – 600.
A pattern of ions was interpreted as
the partial amino acid sequence
Gly-Tyr-Val with a residual mass of
227 Da attached to the glycine resi-
due. The presence of additional ions
FIGURE 1. Electrospray mass spectra defining a mixture of two components with masses of 10,566 and with masses of 28 Da less than this
16,727 Da. Shown is the ESI-MS analysis of a fraction from the reverse-phase HPLC purification of subunits of
bovine mitochondrial complex I. The envelope of multiply charged molecular ions for two components are pattern, characteristic of a- and
labeled A and B, respectively, and correspond to an uncharacterized component with a molecular mass of b-type ion pairs, suggested that the
10,566 Da and the known SGDH subunit (16,725 Da) of complex I, respectively. The peak at 881.4 m/z contains
both the 12⫹ ion for component A and also the 19⫹ ion for component B. Ions corresponding to b334⫹ and pattern derives from b-type ions,
b344⫹ fragment ions from SGDH are labeled with f. and therefore the N-terminal

OCTOBER 27, 2006 • VOLUME 281 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 32725
Subunit Composition of Bovine Mitochondrial Complex I
sequence of the component contains the sequence Gly-Tyr- of SGDH is 10,566.0, which corresponds precisely with that of
Val, and the residual mass of 227 Da is N-terminal to the glycine the unknown component.
(Fig. 2). The analysis of another protein ion (961.6 m/z) con- Analysis of the same sample that was analyzed by mass spec-
taining eleven positive charges produced a similar pattern of trometry by Edman degradation gave only the N-terminal
fragment ions and a partial amino acid sequence (data not sequence of subunit SGDH, and the sequence from residue 56
shown). This partial sequence corresponds to residues 58 – 60 onward was not detected. Moreover, the only band detected by
of the SGDH subunit, and the 227-Da residual mass is SDS-PAGE analysis of the same sample was subunit SGDH.
accounted for by Pro-56 and Glu-57. Hence, the 10,566 frag- Therefore, the 10,566-Da component is not a proteolysis prod-
ment has arisen by cleavage of the peptide bond between Ile-55 uct of subunit SGDH, but it appears to have arisen by scission of
and Pro-56. The calculated molecular mass of residues 56 –143 an Ile–Pro bond during electrospray ionization. This proposal
was confirmed by tandem MS by
collision-induced dissociation of a
protein ion (m/z 1116) containing
15 protons from the spectrum of the
intact SGDH subunit (Fig. 3). This
tandem mass spectrum contained
ions that had arisen from the N-ter-
minal region of subunit SGDH and
also other ions that were common
to both the ESI-MS spectrum of the
two components (Fig. 1) and the
tandem MS fragmentation of a pro-
FIGURE 2. Fragment ion spectrum of a 1057.5 (10ⴙ) protein ion from the 10.6-kDa component. A series of tein ion from the 10,566-Da compo-
dominant b- and a-type fragment ions define the amino acid sequence GYV. The N-terminal sequence PE was nent (Fig. 2). A series of quintuply
deduced from the b2 ion of 227.1 Da and the known protein sequence of the SGDH subunit. An incomplete y charged b-type fragments, b33– b42
ion series (y1– 4, y7–9) defining the amino acid sequence HS(PKA)TPDN (residues 135–143) at the C terminus of
subunit SGDH is also labeled, as are a tyrosine immonium ion (Tyr, 136.1 Da) and significant internal fragment (Fig. 3), defined the amino acid
ions PD (residues 141–142), GY (residues 58 –59), and AEL/IEA (residues 50 –52, 101–103/96 –98) (f, left to right). sequence IPVAIGITL correspond-

FIGURE 3. Fragment ion spectrum of a 1116.1 (15ⴙ) protein ion from the SGDH subunit. The expanded region of the spectrum (760 –960 m/z) contains an
annotated series of quintuply charged b fragment ions (also labeled by the symbol ▫ in the main spectrum) that define the amino acid sequence IPVAIGITL
(residues 34 – 42). Triply and quadruply charged versions of these fragments are also marked. Italicized b33, b41, and b42 ions in the expansion denote b-18 ions.
The asterisk highlights b and a ions previously observed from the fragmentation of a 10⫹ ion of the 10,565-Da component (Fig. 2), which define the sequence
(PE)GYV (residues 56 – 60). Also present are multiply charged protein ions A8⫹ to A10⫹ that form part of the series for the 10,565 Da component seen in Fig.
1. The dominant b334⫹ and b344⫹ ions at 971.2 and 999.5 m/z are also present in the MS spectra in Fig. 1, the b34 ion arising from a fragmentation at an Ile–Pro
bond. Other significant ions are labeled, including doubly charged b10 – b12 ions defining the sequence IK (residues 11–12) from SGDH, y3 and y7 ions from
the C terminus of SGDH and internal fragment ions PV or VP (residues 35–36/60 – 61), GIP or PVA (residues 33–35/35–37), and AEL/AEI/ELA/LAE (from residues
50 –52, 101–103/53–55/51–53/52–54, respectively) (f, left to right).

32726 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 • OCTOBER 27, 2006
 Subunit Composition of Bovine Mitochondrial Complex I
ing to residues 32– 42 of subunit SGDH. The dipeptide Ile-Lys
(residues 10 –11 of subunit SGDH) was defined by the doubly
charged fragment ions b10 – b12. Several peaks (Fig. 3, denoted
as A) corresponded to multiply charged molecular ions that
defined the 10,566-Da component. Other fragment ions,
labeled b2– b5 (Fig. 3, asterisks) defined the sequence (PE)GYV.
These ions were common to the tandem mass spectrum of
10,566 protein ions shown in Fig. 2. Other prominent ions with
masses of 971.2 and 999.5 corresponded to b33 (4⫹) and b34
(4⫹) ions produced by cleavage of SGDH in a second Ile-Pro
sequence at residues 34 –35. These ions were observed also at
lower abundance in the ESI mass spectra (Fig. 1). A molecular
mass corresponding to residues 1–55 of subunit SGDH with a
predicted molecular mass of 6178.4 Da was not observed. Pre-
sumably, it had broken down into smaller fragments.
Protein Cleavage during Electrospray Ionization—The
absence of fragmentation of biological macromolecules is a fea-
ture of soft ionization methods that has contributed to their
success (30, 31), and therefore the cleavage of a protein during
electrospray ionization is extremely unusual. The cleavage has
been observed in three different mass spectrometers with dif-
ferent electrospray interfaces. They are 1) a triple quadrupole
mass spectrometer with an electrospray interface, 2) a hybrid
quadrupole time-of-flight instrument with a Z-spray electro-
spray source and a nanoflow interface, and 3) a second triple
quadrupole instrument with a conventional ion-spray interface
or with sample introduced from nanoelectrospray needles with
single stage transfer of ions to high vacuum through a single
orifice. Many thousands of proteins have been analyzed with-
out fragmentation in the same instruments with the same oper-
ating parameters. However, it is known from collision-induced
dissociation of peptides and from experiments, where the cone
voltage is used to induce fragmentation, that bonds involving
proline rupture most readily (32–34).
The Subunit Composition of Bovine Complex I—The experi-
ments described here resolve a long-standing uncertainty about
the subunit composition of bovine complex I. Recently, six of
the seven hydrophobic subunits of the enzyme that are encoded
in mitochondrial DNA have been characterized by mass spec-
trometry.5 Only subunit ND6 remains to be analyzed to com-
plete the protein chemical characterization of the 45 subunits
of the enzyme.
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