(Received for publication, August 23, 1995; and in revised form, September 27, 1995)
From the
The nicotinamide nucleotide transhydrogenase of bovine
mitochondria is a homodimer of monomer M =
109,065. The monomer is composed of three domains, an
NH
-terminal 430-residue-long hydrophilic domain I that
binds NAD(H), a central 400-residue-long hydrophobic domain II that is
largely membrane intercalated and carries the enzyme's proton
channel, and a COOH-terminal 200-residue-long hydrophilic domain III
that binds NADP(H). Domains I and III protrude into the mitochondrial
matrix, where they presumably come together to form the enzyme's
catalytic site. The two-subunit transhydrogenase of Escherichia
coli and the three-subunit transhydrogenase of Rhodospirillum
rubrum have each the same overall tridomain hydropathy profile as
the bovine enzyme. Domain I of the R. rubrum enzyme (the
1 subunit) is water soluble and easily removed from the
chromatophore membranes. We have isolated domain I of the bovine
transhydrogenase after controlled trypsinolysis of the purified enzyme
and have expressed in E. coli and purified therefrom domain
III of this enzyme. This paper shows that an active bidomain
transhydrogenase lacking domain II can be reconstituted by the
combination of purified bovine domains I plus III or R. rubrum domain I plus bovine domain III.
The proton-translocating nicotinamide nucleotide transhydrogenases of mammalian mitochondria and bacteria are integral membrane proteins that catalyze the reaction shown in ,
On-line formulae not verified for accuracy
where n has been determined to be close to unity
(Fisher and Earle, 1982; Rydström et al.,
1987). The bovine mitochondrial enzyme is a homodimer of monomer M = 109,065 (Yamaguchi et al.,
1988; Hatefi and Yamaguchi, 1992). The Escherichia coli and
the Rhodobacter capsulatus transhydrogenases have two unlike
subunits each,
with M
=
54,000
and
with M
=
49,000 (Clarke et al., 1986; Lever et al., 1991), and the former has
been shown to be an
heterotetramer
(Hou et al., 1990). The transhydrogenase of Rhodospirillum
rubrum has three unlike subunits,
1,
2, and
, with
respective molecular masses of 40.3, 14.9, and 47.8 kDa (Yamaguchi and
Hatefi, 1994; Williams et al., 1994). Subunit
1 is water
soluble and easily removed from the R. rubrum chromatophore
membranes; the other two subunits are integral membrane proteins. The
bovine transhydrogenase monomer is composed of three domains, an
NH
-terminal 430-residue-long hydrophilic domain I that
binds NAD(H), a central 400-residue-long hydrophobic domain II that is
largely membrane-intercalated and bears the enzyme's proton
channel, and a COOH-terminal 200-residue-long hydrophilic domain III
that binds NADP(H). In mitochondria, the two nucleotide-binding domains
I and III protrude into the matrix (Yamaguchi and Hatefi, 1991), where
they presumably come together to form the enzyme's catalytic site
in which direct hydride ion transfer takes place between the 4-A
position of NAD(H) and the 4-B position of NADP(H). Together, the two
subunits of the E. coli or the three subunits of the R.
rubrum enzyme also display the same tridomain hydropathy profile,
with similar nucleotide binding characteristics in domains I and III
(Clarke et al., 1986; Yamaguchi and Hatefi, 1993, 1994; Diggle et al., 1995).
The purpose of this communication is to show that unanchored to the membrane-intercalated domain II, the isolated, soluble domains I and III readily interact to reconstitute a structure capable of catalyzing transhydrogenation between NAD(H) and NADP(H).
E. coli cells (1.6 g) were suspended in 32 ml of 50 mM Tris/HCl, pH 7.8, containing 1 mM EDTA and 1 mM
dithiothreitol (buffer C) and disrupted by sonication. The undisrupted
cells were removed by centrifugation at 12,000 rpm for 10 min (Sorvall,
GSA rotor), and the supernatant was centrifuged at 39,000 rpm for 45
min (Beckman 42Ti rotor). Most of the expressed peptide was present in
the supernatant fraction. The supernatant obtained was loaded onto
DEAE-Bio-gel A-agarose column (1.5 14 cm) equilibrated with
buffer C, and peptides were eluted by a linear (0-0.5 M)
gradient of NaCl (total volume, 200 ml). The elution position of domain
III was located by SDS-polyacrylamide gel electrophoresis using
aliquots of the fractions. The fractions containing domain III were
combined and concentrated to
1 ml using a Centricon-10
concentrator. Then the sample was loaded on an Ultrogel AcA34 column
(1.5
68 cm) equilibrated with buffer C containing 0.1 M NaCl, and 0.9-ml fractions were collected. The fractions
containing domain III were combined and concentrated to
1.5 ml by
a Centricon-10 concentrator. Approximately 10 mg of homogeneous domain
III were obtained by this procedure from 500 ml of the culture medium.
Experimental results on the bovine transhydrogenase together
with theoretical considerations have indicated that outward proton
translocation coupled to NADPH NAD transhydrogenation (reverse
of ) is driven via protein conformation change mainly by
the difference in the binding energies of NADPH and NADP (Hatefi and
Yamaguchi, 1992). Recent results from this laboratory have further
supported this view and have provided evidence that the NADP(H)-binding
domain of the E. coli transhydrogenase is in communication
with a region of the membrane-intercalating domain II that appears to
be concerned with proton translocation (Yamaguchi and Hatefi, 1995).
These findings made it particularly desirable to obtain structural
information about the NADP(H)-binding domain III and persuaded us to
express domain III of the bovine enzyme in E. coli. This was
done (see ``Experimental Procedures''), and the expressed
domain III of the bovine transhydrogenase was isolated in good yield.
However, it was necessary to know whether this expressed and isolated
domain III possessed the conformation it has in the native enzyme, even
though preliminary experiments had shown that it binds
[
C]NADPH.
Because hydride ion transfer
between NAD(H) and NADP(H) is direct in the transhydrogenase, one would
expect that the nicotinamide rings of the two nucleotides would be only
a few Angströms apart and the nucleotide-binding
regions of domains I and III would share complementary surfaces and
attractive forces to allow such close approximation. Therefore, it was
thought possible that isolated domains I and III might interact and
catalyze transhydrogenation. As will be seen below, this expectation
proved correct not only for the interaction of isolated domains I and
III of the bovine transhydrogenase but also for cross-interaction of
domain I from the R. rubrum (1 subunit) and the expressed
domain III from the bovine enzymes (Fig. 1).
Figure 1:
Schematic
representation of the tridomain composition of the bovine and the R. rubrum transhydrogenases and the bidomain composition of
the reconstituted transhydrogenases. The bovine enzyme is a homodimer
in which domain I is hydrophilic and binds NAD(H), domain II is
hydrophobic and carries the enzyme's proton channel, and domain
III is hydrophilic and binds NADP(H) (Hatefi and Yamaguchi, 1992;
Yamaguchi and Hatefi, 1993). The R. rubrum transhydrogenase
has three subunits, 1,
2, and
, of which the
1
subunit (domain I) is water soluble, is dimeric as isolated, and binds
NAD(H). The hydrophobic regions of the
2 and
subunits,
together comprising domain II, are hatched. The reconstituted
bovine domains I plus III and the cross-reconstituted R. rubrum domain I (heavy-lined rectangle) plus bovine domain III
lack the hydrophobic domain II as shown.
Fig. 2shows on SDS-polyacrylamide gel the purified domain I
of the bovine transhydrogenase (lane A), domain III of the
bovine enzyme expressed in E. coli (lane B), and
domain I (1 subunit) of the R. rubrum transhydrogenase (lane C). Domain I of the bovine enzyme was isolated after
controlled digestion of the purified enzyme with trypsin. As was shown
previously (Yamaguchi et al., 1990), trypsin cleaves the
bovine enzyme at Lys
-Thr
as well as at
Lys
-Thr
, resulting in two fragments that
copurify with respective molecular mass values of 41.5 and 43 kDa. The
molecular masses of the bovine domain III and the R. rubrum
1 subunit are 20 and 40.3 kDa, respectively, but the former
displays on SDS-gels an anomalous mobility corresponding to an
molecular mass of about 29 kDa.
Figure 2: SDS-polyacrylamide gel electrophoresis of the preparations of domains I and III used for reconstitution. Lane A, domain I of the bovine transhydrogenase isolated after controlled proteolysis of the purified enzyme (Yamaguchi et al., 1990). This preparation contains two peptides of molecular masses 43 and 41.5 kDa. Lane B, domain III of the bovine transhydrogenase expressed in E. coli and isolated therefrom. Lane C, domain I of R. rubrum transhydrogenase isolated from chromatophore membranes. SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli(1970), and to each lane 1 µg of the preparation indicated was added. The gels were stained with Coomassie Blue and destained in 10% acetic acid. The lane to the extreme left shows protein standards with their molecular masses indicated in kDa.
Data for reconstitution of
transhydrogenase activity involving the 20-kDa domain III of the bovine
enzyme with domain I from the same source (43 and 41.5 kDa) or from R. rubrum (1 subunit, 40.3 kDa) are shown in Table 1. It is seen that reconstitution of NADPH
AcPyAD
transhydrogenation activity was much more effective between domain I
from the R. rubrum and domain III from the bovine enzymes than
between these two domains from the latter source and that each domain
added alone to the assay medium exhibited little or no activity. In
these experiments, the protein fragments were placed on a glass rod and
added together to the assay mixture. Preincubation of the fragments
together at high concentration or their separate addition to the assay
medium did not result in very different activities. The effect of pH on
the rate of NADPH
AcPyAD transhydrogenation catalyzed by R.
rubrum domain I plus bovine domain III was similar to that of the
intact enzymes. The activity increased slightly in going from pH 8.5 to
7.5 and by >1.5-fold in going from pH 7.5 to 6.5.
Table 1also shows data for the transhydrogenation reactions
NADH AcPyADP and NADH
AcPyAD as catalyzed by the R.
rubrum domain I (
1 subunit) plus the bovine domain III. It is
seen that relative to the rate of NADPH
AcPyAD reaction, the
rate of the forward transhydrogenation from NADH to AcPyADP is also
quite substantial. In the intact enzyme, this reaction is very much
slower than NADPH
AcPyAD transhydrogenation and is accelerated
about 10-fold in the presence of a proton motive force. The fact that
the rates of the forward and reverse transhydrogenation are not very
different as catalyzed by the protein fragments lacking the
enzyme's proton translocation domain II may indicate that in the
intact enzyme this domain inhibits NADH
AcPyADP
transhydrogenation in the absence of a proton motive force. The last
entry in Table 1shows transhydrogenation from NADH to AcPyAD, an
activity that the intact enzyme does not exhibit unless under special
conditions in the presence of NADP(H) (Wu et al., 1981; Fisher
and Earle, 1982; Hutton et al., 1994). However, we have
observed that the E. coli transhydrogenase mutant
H91K
also catalyzes NADH to AcPyAD transhydrogenation in the absence of
added NADP(H). (
)Glavas et al.(1995) state in a
recent paper that their
H91K mutant contains bound NADP, but the
data were not presented.
In the reaction NADPH AcPyAD
catalyzed by the R. rubrum domain I (
1 subunit) plus the
expressed bovine domain III, apparent K
values, as
determined from Lineweaver-Burk double reciprocal plots, were 10.9
µM for AcPyAD and 3.2 µM for NADPH. The
respective values for the R. rubrum and the bovine
transhydrogenases are 26 and 20 µM (this study and
Yamaguchi et al., 1990). In the E. coli enzyme
mutation of Asp
to Ile, which inhibits proton
translocation by 90%, lowered the apparent K
by 3.5-fold and K
by 3.3-fold
(Yamaguchi and Hatefi, 1995). It is possible that the absence of the
membrane-intercalating domain II in the system reconstituted from
domains I and III has a similar effect on the affinity of domain III
for NADPH.
Table 2contains data on the inhibitory effects of
FSBA and NEM. In the bovine transhydrogenase, FSBA modifies Tyr in the NAD(H)-binding domain I and very slowly modifies
Tyr
in the NADP(H)-binding domain III (Wakabayashi and
Hatefi, 1987). As seen in Table 2, pretreatment with FSBA had
only a small inhibitory effect on the R. rubrum
1 subunit
but nearly completely inactivated the bovine domain III. In five
transhydrogenases whose predicted amino acid sequences are known, the
residue corresponding to the bovine Tyr
is conserved, and
in the R. rubrum
1 subunit this conserved Tyr is in a
glycine-rich region 33 residues downstream of a
fold
and is flanked by residues GGYAKEM, which are the same in the
bovine enzyme (Yamaguchi and Hatefi, 1994). It is therefore surprising
that in the bovine transhydrogenase Tyr
readily reacts
with FSBA, but in the R. rubrum
1 subunit the
corresponding Tyr does not.
The data for the effect of NEM were the
same as would be expected from the behavior of the bovine enzyme toward
NEM, in which this reagent alkylates Cys in domain III.
(Yamaguchi and Hatefi, 1989). As seen in Table 2, NEM had no
inhibitory effect on the R. rubrum
1 subunit but
inhibited the bovine domain III. Also, similar to the intact bovine
enzyme, addition of NADPH but not NADH stimulated the NEM inhibition.
In the bovine transhydrogenase, it was shown that the reason for this
augmentation of the NEM inactivation rate is that in the presence of
NADPH, the effective pK
of Cys
is
lowered from 9.1 to 8.7 (Yamaguchi and Hatefi, 1989). Not shown in Table 2is the inhibitory effect of palmitoyl-CoA
(Rydström, 1972). In the R. rubrum-bovine
cross-reconstituted system of Table 2, palmitoyl-CoA was a
competitive inhibitor with respect to NADPH, with K
= 3.36 µM.
The reconstituted NADPH
AcPyAD transhydrogenase activities shown in Table 1are
considerably less than the activity of the intact enzyme purified from
bovine mitochondria. This may be related to the fact that in the intact
transhydrogenase the attractive forces between domains I and III do not
appear to be strong. In R. rubrum, the
1 subunit (domain
I) is readily washed away from the chromatophore membranes, and in
submitochondrial particles, domains I and III of the bovine enzyme can
each be easily separated once its link to the membrane-intercalated
domain II is severed by controlled proteolysis (Yamaguchi and Hatefi,
1991). Nevertheless, the reconstituted activities shown above indicate
that domains I and III interact physically to form a catalytic unit.
Whether the presence of bound nucleotides strengthens or weakens this
physical association remains to be investigated. What is known is that
in the intact bovine enzyme NADPH binding at domain III makes domain I
more susceptible to attack by dicyclohexylcarbodiimide or trypsin
(Phelps and Hatefi, 1984; Yamaguchi et al., 1990).
The
transhydrogenases of E. coli and bovine mitochondria, whose
amino acid sequences are known and have been purified in the presence
of detergents, are somewhat unstable in the isolated state and tend to
aggregate when concentrated. These problems complicate attempts at
crystallization for structure studies. By comparison, the purified
domain I of the R. rubrum and the expressed and purified
domain III of the bovine enzymes are water soluble and relatively
stable. Furthermore, the R. rubrum domain I (1 subunit)
has been expressed in E. coli and can also be obtained in high
yield (Diggle et al., 1995). Therefore, these preparations
provide an opportunity for structure studies. It may even be possible
to reconstitute and co-crystallize them and solve the structure of the
catalytic sector of the transhydrogenase.