Manipulations in the peripheral stalk of the Saccharomyces cerevisiae F1F0-ATP synthase

The Saccharomyces cerevisiae F(1)F(0)-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F(1). In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F(1)F(0)-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F(1)F(0)-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.     

Genetic complementation between mutant b subunits in F1F0 ATP synthase

In Escherichia coli, a parallel homodimer of identical b subunits constitutes the peripheral stalk of F(1)F(0) ATP synthase. Although the two b subunits have long been viewed as a single functional unit, the asymmetric nature of the enzyme complex suggested that the functional roles of each b subunit should not necessarily be considered equivalent. Previous mutagenesis studies of the peripheral stalk suffered from the fact that mutations in the uncF(b) gene affected both of the b subunits. We developed a system to express and study F(1)F(0) ATP synthase complexes containing two different b subunits. Two mutations already known to inactivate the F(1)F(0) ATP synthase complex have been studied using this experimental system. An evolutionarily conserved arginine, b(Arg-36), was known to be crucial for F(1)F(0) ATP synthase function, and the last four C-terminal amino acids had been shown to be important for enzyme assembly. Experiments expressing one of the mutants with a wild type b subunit demonstrated the presence of heterodimers in F(1)F(0) ATP synthase complexes. Activity assays suggested that the heterodimeric F(1)F(0) complexes were functional. When the two defective b subunits were expressed together and in the absence of any wild type b subunit, an active F(1)F(0) ATP synthase complex was assembled. This mutual complementation between fully defective b subunits indicated that each of the two b subunits makes a unique contribution to the functions of the peripheral stalk, such that one mutant b subunit is making up for what the other is lacking.     

Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics.

A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin. The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant. The insertion led to the production of a truncated gamma subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal gamma subunit. A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes. Removal of the genes coding for the beta and epsilon subunits abolished antibiotic resistance coded by the mutant plasmid. The relationship between antibiotic resistance and the gamma subunit was investigated by testing the antibiotic resistance of plasmids carrying various combinations of unc genes. The presence of genes for the F0 portion of the ATP synthase in the presence or absence of genes for the gamma subunit was not sufficient to cause antibiotic resistance. alpha, beta, and truncated gamma subunits were detected on washed membranes of the mutant by immunoblotting. The first 247 amino acid residues of the gamma subunit may be sufficient to allow its association with other F1 subunits in such a way that the proton gate of F0 is held open by the mutant F1.     

Sec/SRP requirements and energetics of membrane insertion of subunits a, b, and c of the Escherichia coli F1F0 ATP synthase

Previously, the role of YidC in the membrane protein biogenesis of the F(0) sector of the Escherichia coli F(1)F(0) ATP synthase was investigated. Whereas subunits a and c of the F(1)F(0) ATP synthase were strictly dependent on YidC for membrane insertion, subunit b required YidC for efficient insertion (Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A., and Dalbey, R. E. (2003) Biochemistry 42, 10537-10544). In this paper, we investigated other protein components and energetics that are required in the membrane protein assembly of the F(0) sector subunits. We show here that the Sec translocase and the signal recognition particle (SRP) pathway are required for membrane insertion of subunits a and b. In contrast, subunit c required neither the Sec machinery nor the SRP pathway for insertion. While the proton motive force was not required for insertion of subunits b and c, it was required for translocation of the negatively charged periplasmic NH(2)-terminal tail of subunit a, whereas periplasmic loop 2 of subunit a could insert in a proton motive force-independent manner. Taken together, the in vivo data suggest that subunits a and b are inserted by the Sec/SRP pathway with the help of YidC, and subunit c is integrated into the membrane by the novel YidC pathway.     

The molecular neighborhood of subunit 8 of yeast mitochondrial F1F0-ATP synthase probed by cysteine scanning mutagenesis and chemical modification

The detailed membrane topography and neighboring polypeptides of subunit 8 in yeast mitochondrial ATP synthase have been determined using a combination of cysteine scanning mutagenesis and chemical modification. 46 single cysteine substitution mutants encompassing the length of the subunit 8 protein were constructed by site-directed mutagenesis. Expression of each cysteine variant in yeast lacking endogenous subunit 8 restored respiratory phenotype to cells and had little measurable effect on ATP hydrolase function. The exposure of each introduced cysteine residue to the aqueous environment was assessed in isolated mitochondria using the fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein 5-maleimide in osmotically lysed mitochondria and are thus extrinsic to the lipid bilayer, indicating a 21-amino acid transmembrane span. The C-terminal region of subunit 8 was partially occluded by other ATP synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in maintaining the stability of the F(1)-F(0) interaction. Cross-linking using heterobifunctional reagents revealed the proximity of subunit 8 to subunits b, d, and f in the matrix and to subunits b, f, and 6 in the intermembrane space. A disulfide bridge was also formed between subunit 8(F7C) or (M10C) and residue Cys-23 of subunit 6, demonstrating a close interaction between these two hydrophobic membrane subunits and confirming the location of the N termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial F(1)F(0)-ATP synthase.     

F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d.

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the alpha subunit N-terminus is essential for the correct, functional connection of F1 to F0. The alpha-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit alpha, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits gamma and delta which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit alpha, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the gamma subunit uncovered, surround the gamma and the delta subunits located in the central stalk.     

Application of rigid body mechanics to theoretical description of rotation within F0F1-ATP synthase

ATP synthase catalyses the formation of ATP from ADP and P(i) and is powered by the diffusion of protons throughout membranes down the proton electrochemical gradient. The protein consists of a water-soluble F(1) and a transmembrane F(0) proton transporter part. It was previously shown that the ring of membrane subunits rotates past a fixed subunit during catalytic cycle of the enzyme. However, many parameters of this movement are still unknown. In the present study the mutual protein movement in the membrane part of F(0)F(1)-ATP syntase has been analysed within the framework of rigid body mechanics. On the base of available experimental data it was shown that electrostatic interaction of two charged amino acids residues is able to supply quite enough energy for the rotation. The initial torque, which caused the rotation, was estimated as 3.7 pN nm and for this pattern the angular movement of c subunits complex could not physically have a period less than 10(-9)s. If membrane viscosity and elastic resistance were taken into account then the time of a whole turnover could rise up to 6.3 x 10(-3)s. It is remarkable that rotation will take place only under condition when the elasticity (Young's) module of the central stalk (gamma subunit and other minor subunits) is less than 5.0 x 10(7)N/m(2). Thus, for generally accepted structural parameters of ATP synthase, two-charge electrostatic interaction model does not permit rotation of the rotor if elastic properties of the central stalk are tougher than mentioned above. In order to explain the rotation under that condition one should either suppose a shorter distance between subunit a and c subunits complex or assume interaction of more than two charged amino acids residues.     

Proton translocation by the F1F0ATPase of Escherichia coli. Mutagenic analysis of the a subunit

Cassette site-directed mutagenesis was employed to generate mutations in the a subunit (uncB (a) gene) of F1F0ATP synthase. Using sequence homology with similar subunits of other F1F0ATP synthases as a guide, 20 mutations were targeted to a region of the a subunit thought to constitute part of the proton translocation mechanism. ATP-driven proton pumping activity is lost with the substitution of lys, ile, val, or glu for arginine 210. Substitution of val, leu, gln, or glu for asparagine 214 does not completely block proton conduction, however, replacement of asparagine 214 with histidine does reduce enzyme activity below that necessary for significant function. Two or three mutations were constructed in each of four nonpolar amino acids, leucine 207, leucine 211, alanine 217, and glycine 218. Certain specific mutations in these positions result in partial loss of F1F0ATP synthase activity, but only the substitution of arginine for alanine 217 reduces ATP-driven proton pumping activity to undetectable levels. It is concluded that of the six amino acids studied, only arginine 210 is an essential component of the proton translocation mechanism. Fractionation of cell-free extracts of a subunit mutation strains generally reveals normal amounts of F1 specifically bound to the particulate fraction. One possible exception is the arginine 210 to isoleucine mutation which results in somewhat elevated levels of free F1 detectable in the soluble fraction. For nearly all a subunit mutations, F1F0-mediated ATP hydrolysis activity remains sensitive to inhibition by dicyclohexylcarbodiimide in spite of the fact that the mutations block proton translocation.     

Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase.

By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.     

Second stalk of ATP synthase. Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis

ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta and two copies of F(o)b subunits. The second stalk is expected to keep the stator subunits from spinning along with the rotor. We isolated a TF(1)-b'(2) complex (alpha(3)beta(3)gammadeltaepsilonb'(2)) of a thermophilic Bacillus PS3, in which b' was a truncated cytoplasmic fragment of F(o)b subunit, and introduced a cysteine at its N terminus (bc'). Association of b'(2) or bc'(2) with TF(1) did not have significant effect on ATPase activity. A disulfide bond between the introduced cysteine of bc' and cysteine 109 of gamma subunit was readily formed, and this cross-link caused inactivation of ATPase. This implies that F(o)b subunit bound to stator subunits of F(1) with enough strength to resist rotation of gamma subunit and to prevent catalysis. Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b'(2) from TF(1).     

Biochemical and electron microscopic characterization of the F1F0 ATP synthase from the hyperthermophilic eubacterium Aquifex aeolicus

The F(1)F(0) ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits alpha, beta, gamma, delta and epsilon of F(1) and subunits a and c of F(0). Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F(1) and F(0). In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the delta subunit.     

Integration of b subunits of unequal lengths into F1F0-ATP synthase

In Escherichia coli the peripheral stalk of F1F0-ATP synthase consists of a parallel dimer of identical b subunits. However, the length of the two b subunits need not be fixed. This led us to ask whether it is possible for two b subunits of unequal length to dimerize in a functional enzyme complex. A two-plasmid expression system has been developed that directs production of b subunits of unequal lengths in the same cell. Two b subunits differing in length have been expressed with either a histidine or V5 epitope tag to facilitate nickel-affinity resin purification (Ni-resin) and Western blot analysis. The epitope tags did not materially affect enzyme function. The system allowed us to determine whether the different b subunits segregate to form homodimers or, conversely, whether a heterodimer consisting of both the shortened and lengthened b subunits can occur in an intact enzyme complex. Experiments expressing different b subunits lengthened and shortened by up to 7 amino acids were detected in the same enzyme complex. The V5-tagged b subunit shortened by 7 amino acids (b Delta 7-V5) was detected in Ni-resin-purified membrane preparations only when coexpressed with a histidine-tagged b subunit in the same cell. The results demonstrate that the enzyme complex can tolerate a size difference between the two b subunits of up to 14 amino acids. Moreover, the experiments demonstrated the feasibility of constructing enzyme complexes with non-identical b subunits that will be valuable for research requiring specific chemical modification of a single b subunit.     

The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.     

The epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases. Structure, arrangement, and role of the epsilon subunit in energy coupling within the complex

Recent studies show that the epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases is a component of the central stalk that links the F(1) and F(0) parts. This subunit interacts with alpha, beta and gamma subunits of F(1) and the c subunit ring of F(0). Along with the gamma subunit, epsilon is a part of the rotor that couples events at the three catalytic sites sequentially with proton translocation through the F(0) part. Structural data on the epsilon subunit when separated from the complex and in situ are reviewed, and the functioning of this polypeptide in coupling within the ATP synthase is considered.     

The structure of the central stalk in bovine F(1)-ATPase at 2.4 A resolution

The central stalk in ATP synthase, made of gamma, delta and epsilon subunits in the mitochondrial enzyme, is the key rotary element in the enzyme's catalytic mechanism. The gamma subunit penetrates the catalytic (alpha beta)(3) domain and protrudes beneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATP synthesis. In other crystals of F(1)-ATPase, the protrusion was disordered, but with crystals of F(1)-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed. The delta and epsilon subunits interact with a Rossmann fold in the gamma subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couples the transmembrane proton motive force to catalysis in the (alpha beta)(3) domain.     

Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit

The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.     

Mitochondrial F(0)F(1) ATP synthase. Subunit regions on the F1 motor shielded by F(0), Functional significance, and evidence for an involvement of the unique F(0) subunit F(6)

Studies reported here were undertaken to gain greater molecular insight into the complex structure of mitochondrial ATP synthase (F(0)F(1)) and its relationship to the enzyme's function and motor-related properties. Significantly, these studies, which employed N-terminal sequence, mass spectral, proteolytic, immunological, and functional analyses, led to the following novel findings. First, at the top of F(1) within F(0)F(1), all six N-terminal regions derived from alpha + beta subunits are shielded, indicating that one or more F(0) subunits forms a "cap." Second, at the bottom of F(1) within F(0)F(1), the N-terminal region of the single delta subunit and the C-terminal regions of all three alpha subunits are shielded also by F(0). Third, and in contrast, part of the gamma subunit located at the bottom of F(1) is already shielded in F(1), indicating that there is a preferential propensity for interaction with other F(1) subunits, most likely delta and epsilon. Fourth, and consistent with the first two conclusions above that specific regions at the top and bottom of F(1) are shielded by F(0), further proteolytic shaving of alpha and beta subunits at these locations eliminates the capacity of F(1) to couple a proton gradient to ATP synthesis. Finally, evidence was obtained that the F(0) subunit called "F(6)," unique to animal ATP synthases, is involved in shielding F(1). The significance of the studies reported here, in relation to current views about ATP synthase structure and function in animal mitochondria, is discussed.     

The second stalk of Escherichia coli ATP synthase

Two stalks link the F(1) and F(0) sectors of ATP synthase. The central stalk contains the gamma and epsilon subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic alpha(3)beta(3) complex. The two b subunits and the delta subunit associate to form b(2)delta, a second, peripheral stalk extending from the membrane up the side of alpha(3)beta(3) and binding to the N-terminal regions of the alpha subunits, which are approx. 125 A from the membrane. This second stalk is essential for binding F(1) to F(0) and is believed to function as a stator during rotational catalysis. In vitro, b(2)delta is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with delta. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one alpha subunit or alphabeta pair. However, the weak interactions between the polypeptides in b(2)delta pose a challenge for the proposed stator function.     

Mutant residues suppressing rho(0)-lethality in Kluyveromyces lactis occur at contact sites between subunits of F(1)-ATPase

Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing rho(0)-lethality are limited to the ATP1, 2 and 3 genes coding for the alpha-, beta- and gamma- subunits of mitochondrial F(1)-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F(1)F(0)-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress rho(0)-lethality. Positioning of the seven amino acids subject to mutation on the bovine F(1)-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F(1) in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F(1)-ATPase in the maintenance of mitochondrial inner membrane potential.     

Functional incorporation of chimeric b subunits into F1Fo ATP synthase

F(1)F(o) ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F(1) sector and its catalytic sites against the movement of the rotor. In Escherichia coli, the peripheral stalk is a homodimer of identical b subunits, but photosynthetic bacteria have open reading frames for two different b-like subunits thought to form heterodimeric b/b' peripheral stalks. Chimeric b subunit genes have been constructed by substituting sequence from the Thermosynechococcus elongatus b and b' genes in the E. coli uncF gene, encoding the b subunit. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2 (Deltab). Although not all of the chimeric subunits were incorporated into F(1)F(o) ATP synthase complexes, plasmids expressing either chimeric b(E39-I86) or b'(E39-I86) were capable of functionally complementing strain KM2 (Deltab). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the b'(E39-D53) or b(L54-I86) subunit, indicating intragenic suppression. In general, the chimeric subunits modeled on the T. elongatus b subunit proved to be more stable than the b' subunit in vitro. Coexpression of the b(E39-I86) and b'(E39-I86) subunits in strain KM2 (Deltab) yielded F(1)F(o) complexes containing heterodimeric peripheral stalks composed of both subunits.