From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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It has been shown that treatment of bovine
mitochondrial complex I (NADH-ubiquinone oxidoreductase) with NADH or
NADPH, but not with NAD or NADP, increases the susceptibility of a
number of subunits to tryptic degradation. This increased
susceptibility involved subunits that contain electron carriers, such
as FMN and iron-sulfur clusters, as well as subunits that lack electron carriers. Results shown elsewhere on changes in the cross-linking pattern of complex I subunits when the enzyme was pretreated with NADH
or NADPH (Belogrudov, G., and Hatefi, Y. (1994)
Biochemistry 33, 4571-4576) also indicated that complex I
undergoes extensive conformation changes when reduced by substrate.
Furthermore, we had previously shown that in submitochondrial particles
the affinity of complex I for NAD increases by 20-fold in electron
transfer from succinate to NAD when the particles are energized by ATP hydrolysis. Together, these results suggest that energy coupling in
complex I may involve protein conformation changes as a key step.
In addition, it has been shown here that treatment of complex I with trypsin in the presence of NADPH, but not NADH or NAD(P), produced from the 39-kDa subunit a 33-kDa degradation product that resisted further hydrolysis. Like the 39-kDa subunit, the 33-kDa product bound to a NADP-agarose affinity column, and could be eluted with a buffer containing NADPH. It is possible that together with the acyl carrier protein of complex I the NADP(H)-binding 39-kDa subunit is involved in intramitochondrial fatty acid synthesis.
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INTRODUCTION |
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Among the enzyme complexes of the mitochondrial electron
transport/oxidative phosphorylation system, complex I (NADH-ubiquinone oxidoreductase) has the most complicated structure and mechanism of
action. The enzyme is composed of 43 subunits (1, 2), and contains
one FMN and 5 EPR-visible iron-sulfur clusters, designated centers N1a,
N1b, N2, N3, and N4 (3). Chemical analysis of iron and sulfide
suggested the possible presence of 8 iron-sulfur clusters per FMN (4).
These results agreed with more recent data on the amino acid sequences
of complex I subunits, in which 8 cysteine motifs were found for
ligating 6 tetranuclear and 2 binuclear iron-sulfur clusters (1,
2).
Early studies showed that bovine complex I could be resolved into three
domains, designated as flavoprotein
(FP),1 iron-sulfur protein
(IP), and hydrophobic protein (HP) (5, 6). This tridomain architecture
appears to be the structural design of complex I from Neurospora
crassa mitochondria (35 subunits) (7), Escherichia
coli plasma membrane (13 subunits) (8, 9), and possibly
Paracoccus denitrificans (14 subunits) (10). Bovine FP is
water-soluble; it is composed of three subunits with molecular masses
of 51, 24, and 9 kDa, and it contains per mol 1 FMN, a tetranuclear
iron-sulfur cluster, and a binuclear iron-sulfur cluster. Data from the
bovine (11), the Paracoccus (12), and the E. coli
(13) enzymes have indicated that the 51-kDa subunit binds NAD(H) and
contains FMN and a tetranuclear cluster (center N3), and the 24-kDa
subunit contains a binuclear cluster (likely center N1a). The bovine IP
is also water-soluble, and is composed of seven major polypeptides with
molecular masses of 75, 49, 30, 18, 15, 13, and 11 kDa, of which the
last two comigrate at Mr ~13,000 upon
SDS-polyacrylamide gel electrophoresis (14, 15). Only the 75-kDa
subunit houses iron-sulfur clusters, and data from the
Paracoccus enzyme have shown that the bacterial analogue of
this subunit contains a tetranuclear, a binuclear (likely center N1b),
and possibly another tetranuclear iron-sulfur cluster (16). In
addition, a 23-kDa subunit of complex I contains 2 cysteine motifs for
housing tetranuclear clusters, and a 20-kDa subunit has a motif of 3 cysteine residues that could ligate another tetranuclear cluster (2).
Electron microscopic studies of membrane crystals of
Neurospora complex I and its peripheral (FP + IP) and
integral (HP) segments have indicated that complex I is L-shaped, with one arm of the L being extramembranous and composed of subunits corresponding to those of bovine FP and IP, and the other arm being
within the membrane and containing the hydrophobic subunits of the
enzyme (17). This hydrophobic domain (HP) contains in both the bovine
and the Neurospora complex I the 7 mitochondrially encoded
subunits of the enzyme (2, 18, 19).
Cross-linking studies on bovine complex I have shown, among other things, that only the 51-kDa subunit of FP (containing FMN and one iron-sulfur cluster) cross-links to only the 75-kDa subunit of IP (the only IP subunit that contains iron-sulfur clusters) (20). The interaction of these two subunits was confirmed by ligand blotting, which also showed that IP binds to the 42-, 39-, 23-, 20-, and 16-kDa subunits of HP (21). In addition, we found that treatment of complex I with NAD(P)H, but not with NAD(P), altered the extent of cross-linking among the FP subunits, between the 51- and the 75-kDa subunits, among the IP subunits, and between the IP and the HP subunits (22).
This report presents data on the effects of NAD(H) and NADP(H) on the degradation of bovine complex I subunits by trypsin. In addition, it shows that in the presence of NADPH, but not NADP or NAD(H), the tryptic digestion of the 39-kDa subunit is arrested after comminution to a 33-kDa product. The significance of these results is discussed in relation to (a) redox-induced conformation changes of complex I as a possible mechanism for energy transduction, and (b) the possible role of certain complex I subunits in intramitochondrial fatty acid synthesis.
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EXPERIMENTAL PROCEDURES |
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Materials-- NAD, NADH, NADP, and NADPH were from Calbiochem. TPCK-treated trypsin was from Sigma. Asolectin was from Associated Concentrates. Rotenone was from S. B. Penick and hexaammineruthenium chloride from Strem Chemicals. Complex I was isolated as described previously (23). NADP-agarose (type I) was prepared by the method of Mosbach et al. (24). Ubiquinone-1 (Q1) was a gift from Eisai Chemical (Tokyo, Japan). Polyclonal antibodies against the purified subunits of complex I were raised and affinity-purified as reported previously (25, 26).
Assay of Enzyme Activities--
NADH-ubiquinone-1 reductase and
NADH-K3Fe(CN)6 reductase activities of complex
I were assayed as described elsewhere (23). NADH-ubiquinone-1 reductase
activity was assayed at 37 °C in a reaction mixture containing 20 mM potassium phosphate, pH 8.0, 0.1 mM
ubiquinone-1, 0.15 mM NADH, 2 mM
NaN3, 200 µg of a suspension of asolectin clarified by
sonication, and 4 µg of complex I. Specific activity was calculated
using an extinction coefficient of 6.22 mM1
cm
1 for NADH at 340 nm.
NADH-Ru(NH3)6Cl3 reductase activity
was assayed at 37 °C in a reaction mixture containing 40 mM Tris-HCl, pH 7.5, 2 mM
Ru(NH3)6Cl3, 0.5 mM
NADH, and 4 µg of complex I (27). NADH oxidation was monitored at 340 nm, and specific activity was calculated as above.
NADH-K3Fe(CN)6 reductase activity was assayed
at 37 °C in a reaction mixture containing 40 mM
Tris-HCl, pH 7.5, 1.6 mM K3Fe(CN)6,
0.15 mM NADH, and 2 µg of complex I. Specific activity was calculated using an extinction coefficient of 1.0 mM
1·cm
1 for ferricyanide at
410 nm.
Treatment of Complex I with Trypsin-- Complex I (2 mg/ml) was digested with TPCK-treated trypsin (trypsin: complex I weight ratio = 1:100) at 30 °C in 50 mM triethanolamine-HCl, pH 8.0, containing 0.25 M sucrose and 0.1% Triton X-100 in the absence or the presence of nucleotides as indicated in the figure legends. At the indicated time intervals, aliquots of the reaction mixture were removed and mixed with 2-fold excess of trypsin inhibitor to stop further digestion. Samples of this mixture were used for assay of the remaining enzyme activities and analysis of the digestion pattern of complex I subuints by SDS-polyacrylamide gel electrophoresis and immunoblotting with affinity-purified, subunit-specific antibodies.
Immunoblotting--
Protein samples were denatured by the
addition of an equal volume of SDS denaturation buffer (125 mM Tris-HCl, pH 6.8, 10% glycerol, 10%
-mercaptoethanol, and 4% SDS) followed by heating in boiling water
for 4 min, then subjected to 12% SDS-polyacrylamide gel
electrophoresis (28). Peptides were transferred to nitrocellulose membranes using a Bio-Rad Mini Trans-Blot apparatus. The
electrophoretic buffer solution contained 20 mM Tris
acetate, pH 8.3, 1 mM EDTA, and 0.2 mM
dithiothreitol. Electrotransfer was performed at 30 V for 1 h.
After peptide transfer, membranes were incubated with 5% skim milk in
a buffer containing 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20 (TBST) for 1 h, and then
for another 1 h with the appropriate affinity-purified antibodies
in TBST containing 1% skim milk. The membranes were washed three times with TBST and incubated for 1 h with anti-rabbit IgG-peroxidase conjugate. The membranes were washed again with TBST four times and
x-ray films were developed with the enhanced chemiluminescence detection system.
Distribution of the Tryptic Fragment of the 39-kDa Subunit in the Supernatant and Pellet Fractions-- Complex I was digested with trypsin for 40 min in the presence of 3 mM NADPH, trypsin inhibitor was added, and the sample was centrifuged in a Beckman Airfuge at 30 p.s.i. for 10 min. Aliquots of the supernatant and pellet fractions obtained were suspended in SDS-denaturation buffer and subjected to SDS-polyacrylamide gel electrophoresis. Peptides were transferred onto nitrocellulose membranes and immunoblotted with affinity-purified antibodies to the to 39-kDa subunit, as described above.
Affinity Chromatography of Trypsin-treated Complex I on NADP-Agarose Column-- Complex I (2 mg/ml) was digested with trypsin in the presence of 3 mM NADPH for 40 min. The supernatant fraction was obtained as described above. After overnight dialysis against 500 ml of 10 mM sodium phosphate, pH 7.0, containing 1 mM dithiothreitol and 0.05% Triton X-100, the supernatant was applied to a column (1.5 × 4 cm) of NADP-agarose. The column was washed with 10 ml of the dialysis buffer, eluted with 5 ml of the dialysis buffer containing 1 mM NADPH, and again with 10 ml of the dialysis buffer. Fractions (1.4 ml) were collected, and 10 µl-aliquots of each fraction was denatured and subjected to SDS-polyacrylamide gel electrophoresis. Peptides were transferred onto nitrocellulose membranes and immunoblotted with affinity-purified antibodies against the 39-kDa subunit.
Protein Sequence Analysis-- Complex I (200 µg of protein), digested with 2 µg of trypsin in the presence of 3 mM NADPH, was subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (29) as described above. The membrane was stained with a mixture of Ponceau S (0.09%) and Coomassie Blue (0.01%) and destained, and the 33-kDa peptide band derived from the 39-kDa subunit of complex I was excised for amino acid sequencing. Sequence analysis of the peptide was performed with an Applied Biosystems model 492 protein sequencer at the protein core facility of this institution.
Other Methods-- The protein concentration was determined by the method of Lowry et al. as modified by Peterson (30) or by the biuret method (31), using bovine serum albumin as a standard.
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RESULTS |
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The preparation of bovine heart complex I used in these studies
reduced Q1 by NADH at a rate of 8.7 µmol
(min·mg)1 at 37 °C. This activity was
95%
inhibited by rotenone. To study the effect of trypsin on the
degradation of complex I subunits in the absence or the presence of
substrates, it was necessary to disperse complex I at 2 mg/ml by the
addition of 0.1% Triton X-100. At the concentration used, Triton X-100
reduced the NADH-Q1 reductase activity of complex I down to
7 µmol (min·mg)
1, and lowered rotenone sensitivity to
86% (Fig. 1). Incubation of this mixture
at 30 °C with trypsin abolished the rotenone sensitivity of
Q1 reduction, as seen in Fig. 1. This indicated that the
degradation of complex I subunits by trypsin allowed the primary
dehydrogenase, FP, to catalyze electron transfer from NADH to
Q1, which is a rapid rotenone-insensitive reaction.
Evidence that the catalytic activity of FP is not destroyed by trypsin
under these experimental conditions is also provided in Fig. 1. It is
seen that the activity of complex I, or its FP moiety, for electron
transfer from NADH to ferricyanide or hexaammineruthenium chloride was
little affected even after 60 min of incubation with trypsin (32, 33).
The SDS-gel pattern of complex I subunits subjected to proteolysis by trypsin (Fig. 2) is consistent with
these results.
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Fig. 2 shows on SDS-polyacrylamide gel the electrophoretic pattern of the subunits of complex I before (lane 1) and after the treatment of complex I with trypsin in the absence (lane 2) and presence of 3 mM each of NAD (lane 3), NADH (lane 4), NADP (lane 5), or NADPH (lane 6). The positions of the subunits identified by immunoblotting with affinity-purified, subunit-specific antibodies are marked on the left in Fig. 2, and the positions of their tryptic products, where possible, on the right in Fig. 2. As seen in lanes 2, 3, and 5, the degradation of complex I subunits by trypsin was essentially the same in the absence and the presence of NAD(P). The 75-kDa subunit was partially degraded, the 51- and the 49-kDa subunits were converted to slightly smaller and slightly faster moving bands, and the 42-, 39-, 30-, 18-, and 9-kDa subunits were largely degraded. The 9-kDa subunit is not discernible in Fig. 2, but its nearly complete degradation within the 40-min incubation time of Fig. 2 was ascertained by immunoblotting. The results of Fig. 2 on the effect of trypsin on the 51-, 24-, and 9-kDa subunits (components of FP) together with the data of Fig. 1 indicate that the curtailed 51-kDa subunit was still capable of catalyzing the dehydrogenation of NADH, and that the 9-kDa subunit was not required for this reaction. The latter point agrees with the fact that the NADH-Q reductases of E. coli (8) and P. denitrificans (10) do not have a subunit analogous to the mitochondrial 9-kDa subunit (20). In addition to the above, other subunits to which we do not have antibodies were also degraded by trypsin, such as the two bands below the 24-kDa subunit and the band above the 18-kDa subunit. The mtDNA-encoded subunits ND1 and ND2 (18) move under the conditions of Fig. 2 gel very close to the 30-kDa band. Immunoblots showed that ND1 was largely and ND2 was partially degraded by trypsin, and the presence of different substrates caused no discernible change on the extent of their digestion by trypsin (data not shown).
In contrast to NAD and NADP, which showed little or no effect on the digestion of complex I subunits by trypsin, NADH (Fig. 2, lane 4) and NADPH (Fig. 2, lane 6) enhanced the trypsin susceptibility of the 75-kDa subunit, and NADPH, but not NADH, stabilized a 33-kDa digestion product of the 39-kDa subunit (see the marker on the right in Fig. 2). The effect of NADH on the tryptic digestion of the 75-kDa subunit is more clearly demonstrated in Fig. 3 (compare lanes 2 and 3 under 75 kDa). This figure also shows that NADH enhanced the tryptic digestion of the 49- and the 30-kDa subunits of IP, and Fig. 4 shows similar data for the 51- and 24-kDa subunits of FP and the 23-kDa subunit of HP. In addition, NADH slightly enhanced the tryptic degradation of the 18-kDa subunit of IP, but had no effect on the digestion of the 13- and the 11-kDa subunits, which move on SDS-polyacrylamide gels as two closely associated bands. In the above cases, NADPH affected the tryptic digestion of complex I subunits in a manner similar to NADH, suggesting that it is substrate reduction of complex I that alters its conformation and makes certain subunits more exposed to attack by trypsin.
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An exception to the above was the unique effect of NADPH on the 39-kDa subunit of complex I. As seen in Fig. 5A, NAD, NADP, and NADH had no particular effect on the tryptic digestion of the 39-kDa subunit, whereas the presence of NADPH (lanes 6 and 7) resulted in the formation of a 33-kDa product that resisted further tryptic digestion. Fig. 5B shows that the accumulation of the 33-kDa stable product increased with the concentration of NADPH in the digestion medium, suggesting that the stabilization of the 33-kDa product against further proteolysis was a consequence of NADPH binding. This possibility agrees with the data of Fearnley and Walker (1), showing that near the NH2 terminus of the 39-kDa subunit of the bovine and the homologous 40-kDa subunit of the N. crassa complex I there is a nucleotide (ADP)-binding motif similar to the NADH-binding sequences of several NAD(H)-linked dehydrogenases, including the 51-kDa subunit of bovine complex I. It was therefore of interest to see whether this nucleotide-binding sequence was still present in the 33-kDa product of the 39-kDa subunit. Accordingly, the 33-kDa band was transferred to polyvinylidene difluoride membranes from SDS-gels of complex I treated with trypsin in the presence of NADPH, then excised and subjected to NH2-terminal sequence analysis. It was found that the first 13 NH2-terminal amino acids of the 39-kDa subunit had been severed by trypsin at the Arg13-Ser14 bond, leaving the nucleotide-binding sequence, which starts downstream of Val20, intact. In this region there are two possible trypsin-susceptible bonds at Arg32-Tyr33 and Arg40-Met41, which were probably protected by the bound NADPH. Very likely the conformation of the NADPH bound 33-kDa polypeptide prevented the hydrolysis of other susceptible bonds by trypsin as well.
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To ascertain that the 33-kDa digestion product of the 39-kDa subunit was indeed capable of binding NADP(H), complex I was incubated with trypsin for 40 min in the presence of 3 mM NADPH; further digestion was arrested by the addition of trypsin inhibitor, and the mixture was subjected to centrifugation. SDS-polyacrylamide gel electrophoresis and immunoblotting with affinity-purified antibodies against the 39-kDa subunit showed that both the 39-kDa subunit and its 33-kDa tryptic fragment were partitioned between the pellet and the supernatant (Fig. 6A). The supernatant was then dialyzed and chromatographed on a NADP-agarose column, and, after appropriate washes with the dialysis buffer, the column was eluted with the same buffer containing 1 mM NADPH. Both the 39-kDa and its 33-kDa tryptic fragment were eluted under these conditions (Fig. 6B), indicating that both polypeptides were capable of binding to NADP-agarose and of elution by a buffer containing NADPH. These results together with those of Fig. 5A indicate that the conformations of the NADP-bound and the NADPH-bound 39-kDa subunit are different, and only the latter conformation is largely resistant to tryptic digestion.
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DISCUSSION |
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As explained in the Introduction, our previous studies had shown that the cross-linking pattern of complex I subunits changes when the enzyme is reduced with NADH or NADPH. These changes involved the proximity of subunits within FP and IP as well as between the FP and IP subunits and between the IP and HP subunits (22). In the present work, the data shown above also indicate that reduction of complex I by either NADH or NADPH increases the susceptibility to trypsin digestion of a number of the subunits found in the FP, IP, and HP domains of complex I. These extensive structural changes effected by reduction of complex I may be considered an expected consequence of the change in the Coulombic forces within the enzyme when it is reduced, or may be considered of greater significance and indicative of redox-linked conformation change of the enzyme as a coupling mechanism for proton translocation.
A large number of theoretical mechanisms have been proposed for proton translocation by complex I, involving NADH, FMN, and/or ubiquinone as the proton carriers (34-39). Many of these proposed theories were inspired by the Q-cycle hypothesis of electron transfer and proton translocation by complex III (ubiquinol-cytochrome c oxidoreductase) (40). There is otherwise no experimental support for any of these proposed mechanisms of proton translocation by complex I, and our recent studies have revealed several important features of complex III that cannot be reconciled with the Q-cycle hypothesis (41, 42). Furthermore, a Q-cycle type mechanism cannot be considered for the sodium-translocating NADH-Q reductase (43) with the flavin or the quinone being the Na+-transporting agent.
By contrast, there is clear evidence in several systems that energy
transduction involves conformation change as a key step. In
bacteriorhodopsin light energy drives the conformation change of
all-trans- to 13-cis-retinal and the consequent
loss of a proton from the protonated Schiff base that connects
all-trans-retinal to Lys216 of the protein (44).
In ATP synthase and the nicotinamide nucleotide transhydrogenase
substrate binding energy (ATP in the former, NADPH in the latter)
drives protein conformation changes that result in proton translocation
(45-47). Undoubtedly substrate binding energy is also utilized via
protein conformation change for proton translocation by the vacuolar
ATPases (48). Therefore, it is possible that in complex I (as it is
also clearly the case in cytochrome oxidase) there are no specific
proton carriers. Rather, the redox energy is coupled to protein
conformation changes that result in pKa changes of
appropriate amino acid residues and the consequent release and uptake
of protons across the membrane-intercalated domain of the enzyme. This
notion is supported by our previous finding that in electron transfer
from succinate to NAD, as catalyzed by submitochondrial particles, the
affinity of complex I for NAD increases 20-fold when the particles
are energized by ATP hydrolysis (49).
Another finding of interest described above is the effect of NADPH on the tryptic degradation of the 39-kDa subunit of complex I. In this case, NADPH appears to bind to the 39-kDa subunit and results in the formation of a 33-kDa fragment, which resists further trypsinolysis. Amino-terminal sequencing of the 33-kDa fragment showed that it had lost the first 13 amino acid residues of the 39-kDa subunit, and obviously a larger COOH-terminal segment to make it ~6 kDa smaller than its parent polypeptide. However, it had retained the putative nucleotide-binding region downstream of Val20, which made it capable of binding to NADP-agarose. Presumably, therefore, the 33-kDa fragment was also capable of binding NADPH and assuming a conformation resistant to trypsin attack. These observations pose a question regarding the role of this 39-kDa NADP(H)-binding, but not NAD(H)-binding, subunit of complex I. It was shown in 1973 by this laboratory that complex I was also capable of very slow NADPH oxidation, which rate was accelerated considerably at pH < 6.5 (50). Similar results were obtained with the FP fraction of complex I (51). These as well as EPR studies of NADPH-reduced complex I allowed the conclusion that NADH and NADPH oxidation involved the same complex I subunits and redox centers (52). This conclusion agreed with our subsequent determination by radioimmunoassay that per mol of complex I there is 1 mol each of the 51-, 24-, and 9-kDa subunits of FP and the 75-, 49-, 30-, and 18-kDa subunits of IP (21). These data do not agree, however, with the suggestion of Albracht and de Jong (53) that complex I contains separate primary flavoproteins for the dehydrogenation of NADH and NADPH. One may ask, therefore, whether the 39-kDa subunit, which does not appear to bind NADH, is the specific NADPH dehydrogenase suggested by Albracht and de Jong (53). We do not believe so, because all the flavin of complex I is associated with the 51-kDa subunit of FP. Rather, we think that the 39-kDa subunit might be involved together with the acyl carrier protein subunit of complex I in fatty acid synthesis (54-58).
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ACKNOWLEDGEMENT |
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We thank C. Munoz for the preparation of bovine heart mitochondria and extracts.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant DK08126.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This is Publication No. 11037-MEM from The Scripps Research Institute, La Jolla, CA.
Present address: Dept. of Vascular Biology, The Scripps Research
Institute, La Jolla, CA 92037.
§ To whom correspondence should be addressed. Tel.: 619-784-2054; Fax: 619-784-2054.
1 The abbreviations used are: FP, flavoprotein; IP, iron-sulfur protein; HP, hydrophobic protein; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; Q1, ubiquinone-1.
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REFERENCES |
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