Coupling between catalytic sites and the proton channel in F1F0-type ATPases.

F1F0-type ATPases catalyse both ATP-driven proton translocation and proton-gradient-driven ATP synthesis. Recent cryoelectronmicroscopy and low-resolution X-ray studies provide a first glimpse at the structure of this complicated membrane-bound enzyme. The F1 part is roughly globular and linked to the membrane-intercalated F0 part by a narrow stalk domain, which contains the gamma-, delta- and epsilon-subunits along with domains of the b-subunit of the F0 part. Here, we review evidence that conformational and positional changes in the gamma- and epsilon-subunits provide the coupling between catalytic sites and proton translocation within the F1F0 complex.

Arrangement of the epsilon subunit in the Escherichia coli ATP synthase from the reactivity of cysteine residues introduced at different positions in this subunit.

ECF1F0 has been purified from three mutants in which a Cys has been incorporated by site-directed mutagenesis in the epsilon subunit: these mutants are epsilon S10C, epsilon H38C and epsilon S108C, respectively. ECF1F0 from the mutant epsilon S10C had a 2-fold higher activity than wild-type enzyme, due to altered association of the epsilon subunit with the rest of the complex, and yet showed normal proton pumping function. The other two mutants had ATPase activities similar to wild-type enzyme. The introduced Cys was exposed for reaction with maleimides in epsilon S10C and epsilon S108C. In epsilon H38C, the introduced Cys reacted readily with N-ethylmaleimide in isolated ECF1, but was unavailable for reaction with this or other maleimides in ECF1F0. When this Cys at position 38 in the epsilon subunit was reacted with various maleimides in isolated ECF1 and then the ECF1 bound back to F0, the interaction between the two parts was perturbed. While ECF1F0 reconstituted with unmodified ECF1 functioned normally, enzyme with maleimide-reacted Cys-38 showed much reduced proton pumping, had only around 50% of the DCCD inhibition of unmodified or wild-type enzyme, and had a much higher LDAO activation (as much as 8.3-fold, c.f. 4-fold for wild type). Nucleotide-dependent conformational changes have been observed previously, in studies of ECF1 from the mutants epsilon S10C and epsilon S108C. Identical nucleotide-dependent structural changes were observed in cross-linking experiments with tetrafluorophenylazide maleimides when the intact ECF1F0 from these mutants was examined. Taken together, the Cys reactivity data and cross-linking results provide the orientation of the epsilon subunit in the enzyme complex.

Effect of lipid fluidity upon the activity and structure of the 39 kDa porin from Enterobacter cloacae 908S.

The 39 kDa porin from Enterobacter cloacae 908S was isolated in a lipopolysaccharide-free form using the non-ionic detergent, octylpentaoxyethylene, and reconstituted into vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC), respectively. Porin activity, measured by the rate of hydrolysis of the lipid-impermeant beta-lactam cephazoline by entrapped lactamase, could be demonstrated for porin-DMPC but not for porin-DOPC vesicles, and for the former was significantly lower in the gel than in the liquid-crystalline phase. The fluorescence changes are thought to arise from lipid phase-induced structural/dynamic changes of the porin structure.

Trapping of conformations of the Escherichia coli F1 ATPase by disulfide bond formation. A state of the enzyme with all three catalytic sites of equal and low affinity for nucleotides.

A mutant of Escherichia coli F1F0-ATPase, alphaS411C/betaY331W/betaE381C/gammaC87S, has been generated. CuCl2 treatment of this mutant led to cross-linking between alpha and beta subunits in yields of up to 90%. This cross-linking across non-catalytic site interfaces inhibited ATP hydrolysis activity. In the absence of cross-linking, MgATP bound in catalytic sites of the mutant with three different affinities of 0.1 microM, 6 microM and 60 microM, respectively, values that are comparable to wild-type. For MgADP, there was one tight site (0.34 microM) and two sites of lower affinity (each 27 microM), again comparable to wild-type enzyme. After cross-linking all three catalytic sites bound MgATP or MgADP with the same relatively low affinity (approximately 60 microM). Thus cross-linking fixed all three catalytic sites in the same conformation. Trypsin cleavage experiments showed that cross-linking fixed the epsilon subunit in the ATP+EDTA conformation.

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact.

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.

Yeast cytochrome c oxidase subunit VII is essential for assembly of an active enzyme. Cloning, sequencing, and characterization of the nuclear-encoded gene.

The gene COX VII coding for yeast cytochrome c oxidase subunit VII has been cloned by a two-step procedure. Two degenerate oligonucleotides corresponding to amino- and carboxyl-terminal protein segments were used in a polymerase chain reaction for the amplification of a major portion of subunit VII (residues 1-52), which was then used for the cloning of complete COX VII. From the nucleotide sequence, an additional amino-terminal and two additional carboxyl-terminal amino acids are predicted as compared with the described primary sequence (Power, S. D., Lochrie, M. A., and Poyton, R. O. (1986) J. Biol. Chem. 261, 9206-9209). Beside subunit VIIa the subunit described here is the only nuclear encoded subunit of cytochrome c oxidase in yeast without a leader sequence. COX VII exists as a single copy per haploid genome as shown by Southern blot and gene disruption. Null mutants produced by gene disruption at the COX VII locus were respiratory-deficient. No cytochrome c oxidase activity was detectable nor was there an assembly of the oxidase complex.

Cross-linking of the gamma subunit of the Escherichia coli ATPase (ECF1) via cysteines introduced by site-directed mutagenesis.

The gamma subunit of the Escherichia coli F1 ATPase (ECF1) has been altered by site-directed mutagenesis to create five different mutants, gamma-S8C, gamma-S81C, gamma-T106C, gamma-S179C, and gamma-V286C, respectively. ECF1 isolated from four of these mutants had ATPase activities similar to that of a wild-type isogenic strain used as a control, the exception was enzyme isolated from mutant gamma-S81C, which had an ATPase activity of around 70-80% of the wild type. ECF1 isolated from each of the various mutants was reacted with N-(4-(7-(diethylamino)-4-methylcoumarin-3-yl))maleimide (CM). The fluorescent reagent was incorporated into Cys residues placed at positions 8, 106, 179, and 286, but not at 81, indicating which of these Cys residues are on the surface of the gamma subunit in the enzyme complex. Modification of the Cys at position 106 with CM activated the enzyme, and modification of the Cys at position 8 inhibited ATPase activity a small amount; however, modification of Cys at 179 or 286 had no effect on enzyme activity. The four mutants with a reactive Cys were reacted with tetrafluorophenylazide maleimides (TFPAMs), novel photoactivatable cross-linkers. In the mutant gamma-S8C, cross-links were formed between the introduced Cys on the gamma subunit and sites on the beta subunit. This cross-linking between gamma and beta depended on nucleotide conditions under which the photolysis was carried out, with differently migrating cross-linked products being obtained in ATP + EDTA compared with ATP + Mg2+ or ATP + Mg2+ Pi. Cross-linking between beta and gamma inhibited ATPase activity in proportion to the yield of cross-linked product. In the mutant gamma-V286C, cross-links were formed between the introduced Cys on gamma and the alpha subunit which were the same in all nucleotide conditions and which led to inhibition of ATPase activity.

ATP hydrolysis-linked structural changes in the N-terminal part of the gamma subunit of Escherichia coli F1-ATPase examined by cross-linking studies.

A mutant of Escherichia coli F1-ATPase (ECF1) in which the serine residue in position 8 of the gamma subunit has been replaced by a cysteine residue (gamma S8C) has been used to study nucleotide-dependent cross-linking of the gamma subunit to a beta subunit. When examined in the presence of ADP+Mg2+, either supplied directly or as produced during catalytic turnover of ATP+Mg2+, the main cross-linked product generated using the heterobifunctional, photoactivatable, cross-linker tetrafluorophenylazide maleimide-6 had a M(r)(app) of 108,000. When ATP hydrolysis was inhibited, either by cold or by reaction with sodium azide, or when ATP hydrolysis was prevented by the use of adenyl-5'-yl beta,gamma-imidodiphosphate, the main cross-linked products were species with M(r)(app) of 102,000 and 84,000. The nucleotide-dependent switching from one cross-linking pattern to another could only be observed when the epsilon subunit was bound to ECF1; it was not seen in ECF1*, an enzyme preparation missing delta and epsilon subunits, but was observed in preparations selectively depleted of the delta subunit. We conclude that the changes detected in these cross-linking experiments are occurring during the hydrolysis of ATP when the beta-gamma phosphate bond is cleaved and that they are related to the coupling of ATP hydrolysis to proton translocation.

Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1F0-type ATPase.

Mutants of ECF1-ATPase were generated, containing cysteine residues in one or more of the following positions: alphaSer-411, betaGlu-381, and epsilonSer-108, after which disulfide bridges could be created by CuCl2 induced oxidation in high yield between alpha and epsilon, beta and epsilon, alpha and gamma, beta and gamma (endogenous Cys-87), and alpha and beta. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in epsilonSer-108 along with either the DELSEED region of beta (Glu-381) or the homologous region in alpha (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the epsilon subunit. In betaE381C/epsilonS108C the beta-epsilon cross-link was obtained preferentially when Mg2+ and ADP + Pi (addition of MgCl2 + ATP) was present, while the alpha-epsilon cross-link product was strongly favored in the alphaS411C/epsilonS108C mutant in the Mg2+ ATP state (addition of MgCl2 + 5'-adenylyl-beta,gamma-imidodiphosphate). In the triple mutant alphaS411C/betaE381C/epsilonS108C, the epsilon subunit bound to the beta subunit in Mg2+-ADP and to the alpha subunit in Mg2+-ATP, indicating a significant movement of this subunit. The gamma subunit cross-linked to the beta subunit in higher yield in Mg2+-ATP than in Mg2+-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the beta over the alpha subunit.

Cross-linking and electron microscopy studies of the structure and functioning of the Escherichia coli ATP synthase.

ATP synthase, also called F(1)F(o)-ATPase, catalyzes the synthesis of ATP during oxidative phosphorylation. The enzyme is reversible and is able to use ATP to drive a proton gradient for transport purposes. Our work has focused on the enzyme from Escherichia coli (ECF(1)F(o)). We have used a combination of methods to study this enzyme, including electron microscopy and chemical cross-linking. The utility of these two approaches in particular, and the important insights they give into the structure and mechanism of the ATP synthase, are reviewed.

Structural changes in the gamma and epsilon subunits of the Escherichia coli F1F0-type ATPase during energy coupling.

Structural changes in the Escherichia coli ATP synthase (ECF1F0) occur as part of catalysis, cooperativity and energy coupling within the complex. The gamma and epsilon subunits, two major components of the stalk that links the F1 and F0 parts, are intimately involved in conformational coupling that links catalytic site events in the F1 part with proton pumping through the membrane embedded F0 section. Movements of the gamma subunit have been observed by electron microscopy, and by cross-linking and fluorescence studies in which reagents are bound to Cys residues introduced at selected sites by mutagenesis. Conformational changes and shifts of the epsilon subunit related to changes in nucleotide occupancy sites have been followed by similar approaches.

Cross-linking of the delta subunit to one of the three alpha subunits has no effect on functioning, as expected if delta is a part of the stator that links the F1 and F0 parts of the Escherichia coli ATP synthase.

A mutant of the Escherichia coli F1F0-ATPase has been generated (alphaQ2C) in which the glutamine at position 2 of the alpha subunit has been replaced with a cysteine residue. Cu2+ treatment of ECF1 from this mutant cross-linked an alpha subunit to the delta subunit in high yield. Two different sites of disulfide bond formation were involved, i.e. between Cys90 (or the closely spaced Cys47) of alpha with Cys140 of delta, and between Cys2 of alpha and Cys140 of delta. Small amounts of other cross-linked products, including alpha-alpha, delta internal, and alpha-alpha-delta were obtained. In ECF1F0, there was no cross-linking between the intrinsic Cys of alpha and Cys140. Instead, the product generated between Cys2 of alpha and Cys140 of delta was obtained at near 90% yield. Small amounts of alpha-alpha and delta internal were present, and under high Cu2+ concentrations, alpha-alpha-delta was also formed. The ATPase activity of ECF1 and ECF1F0 was not significantly affected by the presence of these cross-links. When Cys140 of delta was first modified with N-ethylmaleimide in ECF1F0, an alpha-delta cross-link was still produced, although in lower yield, between Cys64 of delta and Cys2 of alpha. ATP hydrolysis-linked proton pumping of inner membranes from the mutant alpha2QC was only marginally affected by cross-linking of the alpha to the delta subunit. These results indicate that Cys140 and Cys64 of the delta subunit and Cys2 of the alpha subunit are in close proximity. This places the delta subunit near the top of the alpha-beta hexagon and not in the stalk region. As fixing the delta to the alpha by cross-linking does not greatly impair either the ATPase function of the enzyme, or coupled proton translocation, we argue that the delta subunit forms a portion of the stator linking F1 to F0.

Rotation of a gamma-epsilon subunit domain in the Escherichia coli F1F0-ATP synthase complex. The gamma-epsilon subunits are essentially randomly distributed relative to the alpha3beta3delta domain in the intact complex.

A triple mutant of Escherichia coli F1F0-ATP synthase, alphaQ2C/alphaS411C/epsilonS108C, has been generated for studying movements of the gamma and epsilon subunits during functioning of the enzyme. It includes mutations that allow disulfide bond formation between the Cys at alpha411 and both Cys-87 of gamma and Cys-108 of epsilon, two covalent cross-links that block enzyme function (Aggeler, R., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 13888-13891). A cross-link is also generated between the Cys at alpha2 and Cys-140 of the delta subunit, which has no effect on functioning (Ogilvie, I., Aggeler, R., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 16652-16656). CuCl2 treatment of the mutant alphaQ2C/alphaS411C/epsilonS108C generated five major cross-linked products. These are alpha-gamma-delta, alpha-gamma, alpha-delta-epsilon, alpha-delta, and alpha-epsilon. The ratio of alpha-gamma-delta to the alpha-gamma product was close to 1:2, i.e. in one-third of the ECF1F0 molecules the gamma subunit was attached to the alpha subunit at which the delta subunit is bound. Also, 20% of the epsilon subunit was present as a alpha-delta-epsilon product. With regard to the delta subunit, 30% was in the alpha-gamma-delta, 20% in the alpha-delta-epsilon, and 50% in the alpha-delta products when the cross-linking was done after incubation in ATP + MgCl2. The amounts of these three products were 40, 22, and 38%, respectively, in experiments where Cu2+ was added after preincubation in ATP + Mg2+ + azide. The delta subunit is fixed to, and therefore identifies, one specific alpha subunit (alphadelta). A distribution of the gamma and epsilon subunits, which is essentially random with respect to the alpha subunits, can only be explained by rotation of gamma-epsilon relative to the alpha3beta3 domain in ECF1F0.

Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase.

Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (alpha, beta, gamma, delta, and epsilon), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme. All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the gamma subunit (gamma II, gamma III, and gamma IV), one mAb against delta, and two mAbs against epsilon (epsilon I and epsilon II). These topology data are consistent with the gamma, delta, and epsilon subunits being located at the interface between the F1 and F0 parts of the complex. Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the epsilon subunit tightly bound and acting to inhibit activity and ECF1* in which the delta and epsilon subunits had been removed by organic solvent treatment. ECF1* had an ATPase activity under standard conditions of 93 mumol of ATP hydrolyzed min-1 mg-1, cf. an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the epsilon subunit had been removed by trypsin treatment. The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the alpha subunit, activation by cleavage of gamma, and inhibition with cleavage of the beta subunit. mAbs to the gamma subunit, gamma II and gamma III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the epsilon subunit, as evidenced by density gradient centrifugation experiments. The gamma-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme. The alpha-subunit mAb (alpha I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.

Disulfide bond formation between the COOH-terminal domain of the beta subunits and the gamma and epsilon subunits of the Escherichia coli F1-ATPase. Structural implications and functional consequences.

A set of mutants of the Escherichia coli F1F0-type ATPase has been generated by site-directed mutagenesis as follows: beta E381C, beta S383C, beta E381C/epsilon S108C, and beta S383C/epsilon S108C. Treatment of ECF1 isolated from any of these mutants with CuCl2 induces disulfide bond formation. For the single mutants, beta E381C and beta S383C, a disulfide bond is formed in essentially 100% yield between a beta subunit and the gamma subunit, probably at Cys87 based on the recent structure determination of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). In the double mutants, two disulfide bonds are formed, again in essentially full yield, one between beta and gamma, the other between a beta and the epsilon subunit via Cys108. The same two cross-links are produced with CuCl2 treatment of ECF1F0 isolated from either of the double mutants. These results show that the parts of gamma around residue 87 (a short alpha-helix) and the epsilon subunit interact with different beta subunits. The yield of covalent linkage of beta to gamma is nucleotide dependent and highest in ATP and much lower with ADP in catalytic sites. The yield of covalent linkage of beta to epsilon is also nucleotide dependent but in this case is highest in ADP and much lower in ATP. Disulfide bond formation between either beta and gamma, or beta and epsilon inhibits the ATPase activity of the enzyme in proportion to the yield of the cross-linked product. Chemical modification of the Cys at either position 381 or 383 of the beta subunit inhibits ATPase activity in a manner that appears to be dependent on the size of the modifying reagent. These results are as expected if movements of the catalytic site-containing beta subunits relative to the gamma and epsilon subunits are an essential part of the cooperativity of the enzyme.

Conformational changes in the gamma and epsilon subunits are integral to the functioning of the Escherichia coli H(+)-pumping ATPase (ECF1F0).

ATP synthesis and ATP hydrolysis by F1F0-type ATPases involve conformational changes transmitted from the catalytic site regions to the proton channel, a distance of more than 100 A. Our studies focus attention on the gamma and epsilon subunits that provide a part of the stalk region in the energy-coupling process within the complex. There are conformational changes in the gamma subunit, and translocations of the epsilon unit, linked to nucleotide-binding changes in catalytic sites, which might be expected to alter the interaction of this subunits with c subunits and, hence, be linked to proton translocation.

Reduced expression of outer-membrane proteins in beta-lactam-resistant mutants of Enterobacter cloacae.

Two antibiotic-resistant mutants of Enterobacter cloacae (AZT-R and AMA-R), obtained by selection with aztreonam and carumonam, were studied. Both mutants were resistant to beta-lactam antibiotics. In addition, AMA-R was also resistant to chloramphenicol, trimethoprim and brodimoprim, whereas AZT-R was hypersensitive to these compounds. Cytoplasmic and outer membranes of these bacteria were separated by sucrose density gradient centrifugation. Analysis of the outer membranes using SDS-PAGE showed marked changes in the bands corresponding to the porins (between 35 and 40 kDa). In the two mutants, the 39 kDa band was reduced to approximately 30% of the wild-type and the 36.5 kDa band was absent. Labelling of the outer membranes with the hydrophobic photolabel 3-(trifluoomethyl)- 3-(m-[125I]iodophenyl)diazirine ([125I]TID) enabled the above bands as well as a 28.8 kDa band to be identified as integral membrane proteins, thus supporting the suggestion that they correspond to porins and OmpA protein, respectively. Whereas the changes observed in outer-membrane proteins are assumed to be responsible for resistance to beta-lactam antibiotics, the basis of hypersensitivity of AZT-R to hydrophobic antibiotics remains to be more clearly defined.

Labeling of the ATP synthase of Escherichia coli from the head-group region of the lipid bilayer.

The isolated and membrane-bound forms of the adenosinetriphosphatase of Escherichia coli (ECF1 and ECF1F0, respectively) have been reacted with two lysine-specific reagents, sodium hexadecyl 4-[3H]formylphenyl phosphate (HFPP) and sodium methyl 4-[3H]formylphenyl phosphate (MFPP), and with the photoreactive reagent 1,2-[3H]dipalmitoyl-sn-glycerol 3-[[[(4-azido-2-nitrophenyl)amino]ethyl]-phosphate] (arylazidoPE). HFPP and arylazidoPE are amphipathic molecules, inserting by their hexadecyl moieties (one and two chains, respectively) into the lipid bilayer, with the reactive groups intercalated among the phospholipid head groups. MFPP is the water-soluble analogue of HFPP. The labeling patterns of ECF1F0 obtained with HFPP and arylazidoPE were very similar; in both cases the a and b subunits of the F0 part were the most heavily labeled polypeptides of the complex. Models of subunit a, arranged in six transmembrane helices, place most of the lysines in the head-group region, available for reaction with HFPP. Subunits alpha and beta of the ECF1 part were very poorly labeled in comparison to the a and b subunits, together incorporating only 4% as much HFPP and 7.5% as much arylazidoPE as the two F0 subunits together on a protein mass basis. Trypsin cleavage studies localized any labeling of the alpha subunit by arylazidoPE to the N-terminal 15 residues of this polypeptide. When MFPP was used, the alpha and beta subunits were very much more reacted than the F0 subunits. This implies that most of the mass of the alpha and beta subunits in ECF1F0 is above the membrane and not in contact with the bilayer surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The gammaepsilon-c subunit interface in the ATP synthase of Escherichia coli. cross-linking of the epsilon subunit to the c subunit ring does not impair enzyme function, that of gamma to c subunits leads to uncoupling.

Mutants with a cysteine residue in the gamma subunit at position 207 and the epsilon subunit at position 31 were expressed in combination with a c-dimer construct, which contains a single cysteine at position 42 of the second c subunit. These mutants are called gammaY207C/cc'Q42C and epsilonE31C/cc'Q42C, respectively. Cross-linking of epsilon to the c subunit ring was obtained almost to completion without significant effect on any enzyme function, i.e. ATP hydrolysis, ATP synthesis, and ATP hydrolysis-driven proton translocation were all close to that of wild type. The gamma subunit could also be linked to the c subunit ring in more than 90% yield, but this affected coupling. Thus, ATP hydrolysis was increased 2. 5-fold, ATP synthesis was dramatically decreased, and ATP hydrolysis-driven proton translocation was abolished, as measured by the 9-amino-6-chloro-2-methoxyacridinequenching method. These results for epsilonE31C/cc'Q42C indicate that the c subunit ring rotates with the central stalk element. That the gamma-epsilon cross-linked enzyme retains ATPase activity also argues for a gammaepsilon-c subunit rotor. However, the uncoupling induced by cross-linking of gamma to the c subunit ring points to important conformational changes taking place in the gammaepsilon-c subunit interface during this. Blocking these structural changes by cross-linking leads to a proton leak within the F(0).

Isolation and characterization of the cDNAs encoding two isoforms of subunit CIX of bovine cytochrome c oxidase.

The smallest subunit of bovine cytochrome c oxidase (CIX or VIII in different nomenclatures) occurs in two isoforms, a heart (H) form and a liver (L) form. The cDNAs for both of these forms have been isolated and sequenced. The cDNA for the H form encodes a protein 70 amino acids long with a 24-residue presequence and a mature polypeptide of 46 amino acids; that of liver encodes a protein of 69 amino acids, a 25-residue presequence and a mature polypeptide of 44 amino acids. The leader sequences of the H and L forms are 40% homologous with an abundance of positively charged residues but no negatively charged amino acids. These features are typical of polypeptides targeted to the mitochondrion for processing in the matrix space. The homology of the two isoforms is 52% in the mature subunit with most of the differences occurring in the N-terminal hydrophilic domain of the protein. Evidence has been obtained of polymorphisms of both the H and L forms of the subunit. Protein chemical analyses show that the H isoform is the predominant if not the exclusive form of subunit CIX in heart and skeletal muscle tissue. The L form is the predominant form in liver, kidney, and brain. Northern analyses, using cDNAs to the two forms to screen whole cell RNA preparations, show that the transcript of the H isoform is present in heart and skeletal muscle but not in other tissues examined. The mRNA of the L form was found in brain, kidney, and liver and also in heart and skeletal muscle. These results indicate that the synthesis of the H isoform of CIX is controlled transcriptionally while the L form is under post-transcriptional regulation at least in heart and muscle tissue.