F1 Subunits Research Articles

Cross-reconstitution studies with polypeptides of Escherichia coli and bovine heart mitochondrial F0F1 ATP synthase.

To characterize the role of supernumerary subunits of the mammalian F0F1 ATP synthase, cross-reconstitution of mitochondrial and bacterial F0F1 complexes has been carried out. Escherichia coli F1 (EcF1) can be reconstituted with F1-stripped everted membranes of E. coli (UPEc) and of bovine heart mitochondria (USMP). Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes. Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin-insensitive ATP-hydrolase activity. Subunits of the mammalian F0, in particular F0I-PVP protein, F6 and oligomycin-sensitivity-conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc. Reaction of N,N'-dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP. The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N,N'-dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1. Addition of mitochondrial F0I-PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin. F0I-PVP and OSCP added to UPEc, promoted inhibition by N,N'-dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E. coli F0. The presence of F0I-PVP and OSCP also promoted inhibition by N,N'-dicyclohexylcarbodiimide of the ATP-hydrolase activity of EcF1 or BHF1 reconstituted with UPEc.

Assignment of disulfide bridges in the fusion glycoprotein of Sendai virus.

The mature fusion (F) glycoprotein of the paramyxovirus family consists of two disulfide-linked subunits, the N-terminal F2 and the C-terminal F1 subunits, and contains 10 cysteine residues which are highly conserved at specific positions. The high level of conservation strongly suggests that they are indeed disulfide linked and play important roles in the folding and functioning of the molecule. However, it has not even been clarified which cysteine residues link the F2 and F1 subunits. This report describes our assignment of the disulfide bridges in purified Sendai virus F glycoprotein by fragmentation of the polypeptide and isolation of cystine-containing peptides and determination of their N-terminal sequences. The data demonstrate that all of the 10 cysteine residues participate in disulfide bridges and that Cys-70, the only cysteine in F2, and Cys-199, the most upstream cysteine in F1, form the interchain bond. Of the remaining eight cysteine residues clustered near the transmembrane domain of F1, the specific bridges identified are Cys-338 to Cys-347 and Cys-362 to Cys-370. Although no exact pairings between the subsequent four residues were defined, it seems likely that the most downstream, Cys-424, is linked to Cys-394, Cys-399, or Cys-401. Thus, we conclude that the cysteine-rich domain indeed contributes to the formation of a bunched structure containing at least two tandem cystine loops.

Macromolecular aggregation of F1 and P1 fractions of RU 41740 an immunomodulating compound isolated from Klebsiella pneumoniae, as revealed by size exclusion high-performance liquid chromatography and light scattering detection

RU 41740 isolated from Klebsiella pneumoniae possesses high immunomodulating activity. This immunomodulating agent has been described as composed of two glyeoprotein F1 and P1 fractions. Some characteristics of native RU 41740 and F1 and P1 fractions were investigated using size exclusion high-performance liquid chromatography and light scattering detection. Particle size distribution and apparent molecular mass data suggest that RU 41740 is a macromolecular aggregate of numerous F1 and P1 subunits. Findings suggest an effect of orally administered RU 41740 on the function of Peyer's patch cells.

Suppressor mutations in F1 subunit epsilon recouple ATP-driven H+ translocation in uncoupled Q42E subunit c mutant of Escherichia coli F1F0 ATP synthase.

The Q42E mutation in the polar loop of subunit c of the Escherichia coli F1F0 ATP synthase leads to an uncoupling of H+ translocation through F0 and ATP synthesis/hydrolysis in F1. We have isolated four second-site suppressor mutants in which the coupling defect is corrected. Substitutions for Glu31 in F1 subunit epsilon were found in each suppressor mutant, where the substitutions were E31G, E31V, and E31K (the last being found twice). The different substitutions vary in effectiveness in restoring wild type growth properties in the order epsilon E31G > epsilon E31V > epsilon E31K. Biochemical properties of epsilon E31G/cQ42E and epsilon E31K/cQ42E membranes were compared. In epsilon E31G/cQ42E mutant membranes, ATP-driven H+ translocation by F1F0 and the binding and coupling of F1 to F0 showed a striking pH dependence. Near normal function was observed at pH 7.0, but function was lost at pH 7.8. The function of epsilon E31K/cQ42E membranes was much less affected by changes in pH. Relative to epsilon E31G/cQ42E membranes, the ATP-driven H+ transport function of epsilon E31K/cQ42E membranes was approximately the same at pH 7.5, greater at pH 7.8, and less at pH 7.0. The differences between mutants could be explained if cGlu42 ionized at pH 7.8 with loss of function in epsilon E31G/cQ42E membrane and a similar ionization were compensated for by the positively charged Lys in the epsilon E31K/cQ42E membrane.

Photolabeling of Mitochondrial F1-H+ATPase by 2-Azido[3H]ADP and 8-Azido[3H]ADP Entrapped as Fluorometal Complexes into the Catalytic Sites of the Enzyme

In the presence of ADP and fluorometals, the ATPase activity of the catalytic sector, F1, of beef heart mitochondrial ATPase is strongly inhibited; this inhibition is dependent on the entrapment of ADP-fluoroaluminate complexes into the nucleotide binding sites of F1 [Lunardi, J., Dupuis, A., Garin, J., Issartel, J. P., Michel, L., Chabre, M., & Vignais, P. V. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8958-8962]. We described here the effect of fluoroaluminate on the binding of 2-azido[3H]ADP and 8-azido[3H]ADP to beef heart mitochondrial F1 in the absence and presence of light. When the incubation medium was supplemented with NaF and AlCl3, and maintained in the dark, both 2-azido[3H]ADP and 8-azido[3H]ADP were able to elicit inhibition of F1-ATPase activity, exactly like ADP did. Upon photoirradiation, 2-azido[3H]ADP and 8-azido[3H]ADP bound covalently to F1. Labeling was restricted to the beta subunit of F1, and the same tyrosine residue, beta-Tyr-345, was labeled by either of the photoprobes. This is in contrast with the previous findings that in the absence of fluoroaluminate both the alpha and beta subunits of F1 were photolabeled by 8-azido[3H]ADP, and that two different regions of the beta subunits were labeled, centered on beta-Tyr-345 in the case of 2-azido[3H]ADP [Garin, J., Boulay, F., Issartel, J.P., Lunardi, J., & Vignais, P. V. (1986) Biochemistry 25, 4431-4437] and beta-Tyr-311 that of 8-azido[3H]ADP [Hollemans, M., Runswick, M., Fearnley, I.H., & Walker, J.E. (1983) J. Biol. Chem. 258, 9307-9313].(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and assembly of the F0 proton channel from F0 genes cloned into bacteriophage lambda and integrated into the Escherichia coli chromosome.

The promoter region and the first four genes of the Escherichia coli proton-translocating ATPase (unc) operon, uncIBEF, were cloned into bacteriophage lambda, enabling this region to be recombined into an unc-deleted E. coli chromosome at the lambda att site. The resultant E. coli strain, carrying single-copy F0 genes, was tested for synthesis and assembly of functional F0 proton channels. Membranes isolated from this strain contained all three F0 subunits and were capable of binding purified F1 and reconstituting F1F0-dependent energy coupling activities. The presence of these F0 sectors did not affect cell growth or membrane proton permeability assayed by fluorescence quenching. When compared with wild type membranes, membranes from the single-copy F0 strain contained less a and b subunits. When the single-copy lambda F0 strain was transformed with an F1 plasmid, the cells became phenotypically and biochemically Unc+, with membrane-bound ATPase and ATP synthase activities that were 50-60% of wild type. The results demonstrate that F0 produced from single-copy genes in the absence of F1 is membrane-bound and functional (i.e. reconstitutable) but not freely permeable to protons. The presence of F1 genes and/or subunits during F0 synthesis and assembly both increases the relative amounts of membrane-bound a and b subunits and produces an F0 sector more like that found in wild type cells than is produced from the single-copy F0 genes alone.

Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors

The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes.

Role of the delta subunit in enhancing proton conduction through the F0 of the Escherichia coli F1F0 ATPase.

We studied the effect of the delta subunit of the Escherichia coli F1 ATPase on the proton permeability of the F0 proton channel synthesized and assembled in vivo. Membranes isolated from an unc deletion strain carrying a plasmid containing the genes for the F0 subunits and the delta subunit were significantly more permeable to protons than membranes isolated from the same strain carrying a plasmid containing the genes for the F0 subunits alone. This increased proton permeability could be blocked by treatment with either dicyclohexyl-carbodiimide or purified F1, both of which block proton conduction through the F0. After reconstitution with purified F1 in vitro, both membrane preparations could couple proton pumping to ATP hydrolysis. These results demonstrate that an interaction between the delta subunit and the F0 during synthesis and assembly produces a significant change in the proton permeability of the F0 proton channel.

Defective energy coupling in delta-subunit mutants of Escherichia coli F1F0-ATP synthase.

Membrane vesicles from 13 strains carrying mutations in the C-terminal region of the delta-subunit of Escherichia coli F1F0-ATP synthase were characterized in respect to ATPase activity, ATP-driven proton-pumping, dicyclohexylcarbodiimide sensitivity of ATPase, and oxidative phosphorylation. The salient finding was that energy-coupling between F1 and F0 sectors of the enzyme is impaired by several of the mutations. The delta G150N mutant appeared completely uncoupled in vitro. The data emphasize the role of the C-terminal region of delta-subunit in integration of the proton conduction machinery in F0 with the three F1 catalytic sites. It is suggested that the C-terminal region of delta-subunit, speculatively located in the central region of the alpha 3 beta 3 hexagon, acts functionally at the interface between the helical domain of the stalk and the F1 subunits to relay conformational signals which alter the affinities of the catalytic sites for substrates and products.

Conformational constraints of conserved neutralizing epitopes from a major antigenic area of human respiratory syncytial virus fusion glycoprotein.

To study the conformational requirements of epitopes from a conserved antigenic area (area II) of respiratory syncytial (RS) virus fusion (F) glycoprotein, peptides of increasing length containing amino acids essential for these epitopes were synthesized. The synthetic peptides were tested for binding to a panel of neutralizing monoclonal antibodies (MAbs) for this area as well as to rabbit hyperimmune and human convalescent antisera. Antibody binding was dependent on peptide length; thus, a 61-residue peptide spanning amino acids 215 to 275 of the F1 subunit (peptide F215-275) reacted with more antibodies than a shorter (41-residue) peptide F235-275, and this one with more than the (21-residue) peptide F255-275. Most human convalescent sera contained antibodies that reacted with peptides F215-275 and F235-275 but failed to react with F255-275. The results of antibody binding could be related to the structure adopted by the peptides in solution, as determined by circular dichroism spectroscopy and susceptibility of peptides to trypsin digestion. Pretreatment of peptide F215-275 with SDS abolished reactivity with certain MAbs, supporting the notion that higher order structures were needed for antibody binding. High titre anti-peptide antisera were induced in rabbits inoculated with the peptides; however, these sera failed to react with the native F molecule. In mice, only the largest F215-275 peptide induced an anti-peptide response, but their sera reacted poorly with the native F protein and the animals were not protected against an RS virus challenge. These results illustrate the potential use of synthetic peptides in studies of the F protein physical and antigenic structures as well as the problems in designing synthetic RS virus vaccines.

Structure and sequence comparison of bovine respiratory syncytial virus fusion protein

The fusion (F) proteins of 10 strains of bovine respiratory syncytial virus (BRSV) were compared by radioimmunoprecipitation with fractionation on SDS-polyacrylamide gels. Two different molecular weights (15 kDa and 20 kDa) of the F2 proteins were demonstrated among the BRSV strains tested. To delineate the molecular basis for differences in the molecular weights of F2 subunits among the BRSV strains, the nucleotide sequences of the F genes of FS1 and VC464 strains were determined from cDNA clones. The deduced amino acid sequences were then compared to those of BRSV strains RB94, 391−2 and A51908. The F gene was highly conserved (> 95%) among BRSV strains. Comparison of the deduced F2 amino acid sequences showed that the strain with F2 subunits of 20 kDa had three N-linked glycosylation sites, whereas the strains with F2 subunits of 15 kDa had two N-linked glycosylation sites. Analysis of F2 subunits in their deglycosylated forms indicated that the difference in the molecular weights of the F2 subunits was due to the difference in the extent of glycosylation.

Assembly of the Escherichia coli F1F0 ATPase, a large multimeric membrane‐bound enzyme

The F1F0 proton translocating ATPase of Escherichia coli is a large membrane-bound enzyme complex consisting of more than 20 polypeptides that are encoded by the unc operon. Besides being a system for analysing the enzymology of ATP synthesis and energy coupling, the ATPase is a model system for determining how large oligomeric membrane-bound proteins are synthesized and assembled. The assembly of the ATPase involves differential gene expression and assembly of the subunits within the membrane and with each other. This review discusses the influence of F1 subunits on the assembly and proton permeability of the F0 proton channel, and the possible advantages to assembly of the particular arrangement of genes in the unc operon.

Docking the mitochondrial inhibitor protein IF1 to a membrane receptor different from the F1-ATPase beta subunit.

Monoclonal antibodies reacting with the inhibitor protein (IF1) of the mitochondrial ATPase/ATP synthase complex did not modify the IF1-induced inhibition of soluble F1 ATPase activity. On the contrary, they increased the ATPase activity of inverted electron-transport particles without inducing a significant release of IF1 from these particles. This suggested that IF1 could be linked to a membrane protein when it was not inhibiting the ATPase activity. IF1 antibodies have been used to show that IF1 can bind not only to the beta subunit of F1-ATPase [Klein, G., Satre, M., Dianoux, A. C. & Vignais, P. V. (1981) Biochemistry 20, 1339-1344] but also to a protein present in the inner-mitochondrial membrane. The cross-linking of IF1 to this membrane protein gave a product of M(r) 15000-16000 that migrated differently from IF1 and from the dimer of IF1 using SDS/PAGE. When the cross-linked product was obtained by using a cleavable cross-linking reagent, the complex between IF1 and the docking protein was partly dissociated and free IF1 was recovered. Considering the molecular mass of IF1, this docking protein for IF1 has apparent M(r) 5000-6000. The complex between IF1 and this receptor protein can be detected in low amounts by antibodies against IF1 in the absence of cross-linking reagent. Since this complex remained in the pellet after treatment of the membrane with Triton X-100, it should be a membrane protein. Therefore, IF1 can bind not only to its inhibitory-binding site at the beta subunit of F1, but also to a non inhibitory site which is a membrane protein of approximate M(r) 5000-6000.

The α3β3 and α1β1 Complexes of ATP Synthase

Two catalytic structures of H(+)-motive ATP synthase (Fig. 1), the alpha 3 beta 3 oligomer (M(r) = 319,581) and alpha 1 beta 1 promoter (M(r) = 106,527) (Fig. 2), were isolated using high pressure liquid chromatography (Fig. 3) and polyacrylamide gel electrophoresis (Figs. 4 and 5). These were reconstituted from the alpha and beta subunits of thermophilic F1 (TF1), and the alpha 3 beta 3 oligomer was also crystallized. Common to both F1 and the alpha 3 beta 3 oligomer were the nucleotide specificity, the two Km values, the presence of protomer-oligomer activities, and the one-hit--one-kill phenomenon. A synchrotron experiment on the ATP hydrolysis cycle revealed the dynamic shrinkage and expansion of F1(44) that correspond, respectively, to the ATP-induced association and ADP-induced dissociation of the alpha 3 beta 3 oligomer. The oligomer, like mitochondrial F1 and TF1, exhibited two kinds of ATPase activity: one was cooperative and was inhibited by only one inhibitor per hexamer, and the other was inhibited by three inhibitors per hexamer.

Kinetic studies of ATP synthase: the case for the positional change mechanism.

The mitochondrial ATP synthases shares many structural and kinetic properties with bacterial and chloroplast ATP synthases. These enzymes transduce the energy contained in the membrane's electrochemical proton gradients into the energy required for synthesis of high-energy phosphate bonds. The unusual three-fold symmetry of the hydrophilic domain, F1, of all these synthases is striking. Each F1 has three identical beta subunits and three identical alpha subunits as well as three additional subunits present as single copies. The catalytic site for synthesis is undoubtedly contained in the beta subunit or an alpha, beta interface, and thus each enzyme appears to contain three identical catalytic sites. This review summarizes recent isotopic and kinetic evidence in favour of the concept, originally proposed by Boyer and coworkers, that energy from the proton gradient is exerted not directly for the reaction at the catalytic site, but rather to release product from a single catalytic site. A modification of this binding change hypotheses is favored by recent data which suggest that the binding change is due to a positional change in all three beta subunits relative to the remaining subunits of F1 and F0 and that the vector of rotation is influenced by energy. The positional change, or rotation, appears to be the slow step in the process of catalysis and it is accelerated in all F1F0 ATPases studied by substrate binding and by the proton gradient. However, in the mammalian mitochondrial enzyme, other types of allosteric rate regulation not yet fully elucidated seem important as well.

The alpha beta complexes of ATP synthase: the alpha 3 beta 3 oligomer and alpha 1 beta 1 protomer.

The basic structures of the catalytic portion (F1, alpha 3 beta 3 gamma delta epsilon) of ATP synthase are the alpha 3 beta 3 hexamer (oligomer with cooperativity) and alpha 1 beta 1 heterodimer (protomer). These were reconstituted from the alpha and beta subunits of thermophilic F1 (TF1), and the alpha 3 beta 3 hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F1 and TF1, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.

A model for the catalytic site of F1-ATPase based on analogies to nucleotide-binding domains of known structure.

An updated topological model is constructed for the catalytic nucleotide-binding site of the F1-ATPase. The model is based on analogies to the known structures of the MgATP site on adenylate kinase and the guanine nucleotide sites on elongation factor Tu (Ef-Tu) and the ras p21 protein. Recent studies of these known nucleotide-binding domains have revealed several common functional features and similar alignment of nucleotide in their binding folds, and these are used as a framework for evaluating results of affinity labeling and mutagenesis studies of the beta subunit of F1. Several potentially important residues on beta are noted that have not yet been studied by mutagenesis or affinity labeling.

Role of F0 and F1 subunits in the gating and coupling function of mitochondrial H(+)-ATP synthase. The effect of dithiol reagents.

A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H(+)-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi-ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the gamma subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of gamma subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified gamma subunit was added back. The addition to F0 liposomes of gamma subunit, but not that of alpha and beta subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and gamma subunits are directly involved in the gate of the F0F1 H(+)-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.

Intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage.

The intracellular processing and transport of the respiratory syncytial virus (RSV) fusion (F) glycoprotein was examined by comparing the maturation and stability of wild-type F, uncleaved mutant F and chimeric F glycoproteins expressed by recombinant vaccinia viruses to that of F protein expressed by RSV. One of the recombinant viruses, vF317, expressed F protein (F317) that was processed like the RSV F glycoprotein. F317 was synthesized initially as F0, the uncleaved glycosylated precursor of mature F protein, and formed stable oligomeric structures that were maintained following cleavage of F0 to form the disulphide bond-linked F1 and F2 subunits. Most of the newly synthesized F0 expressed by either RSV or by vF317 was sensitive to treatment with endoglycosidase H (Endo H). Following cleavage of F0, F1 was resistant to Endo H, suggesting that conversion to complex-type sugars, which takes place in the medial Golgi apparatus, occurred simultaneously with or immediately prior to cleavage of F0 into F1 and F2. Another recombinant virus, vF313, synthesized only uncleaved F protein (F313) that comigrated with F0. Uncleaved F313 was expressed as a stable glycosylated protein; however, unlike cleaved F317, its oligosaccharides were not modified to complex forms, as determined from its Endo H sensitivity, and uncleaved F313 did not assemble into stable oligomeric structures. Nucleotide sequence analysis of the cDNA clones encoding F313 and F317 revealed four predicted amino acid sequence differences, none of which were located at the cleavage site. Expression of chimeric F proteins obtained by restriction fragment exchange between the two cDNA clones indicated that two amino acid changes in the F1 domain, located at amino acid residues 301 (Val to Ala) and 447 (Val to Met), resulted in the expression of uncleaved F protein. A change at either of these two amino acid residues, 301 or 447, resulted in the expression of inefficiently cleaved F protein, defining an additional F protein phenotype. Pulse-chase analyses to examine the association of recombinant F glycoproteins with gradient-purified fractionated membranes or with GRP78-BiP, a protein resident in the endoplasmic reticulum (ER) which binds to nascent proteins, revealed that uncleaved F protein (F313) is associated with GRP78-BiP in the ER for a longer time than F317, and little if any F313 was transported to the cell surface. In addition, the uncleaved F protein (F313) was not recognized by a panel of F protein-specific monoclonal antibodies in ELISA or indirect immunofluorescence assays, suggesting that F313 was misfolded and, as a result, not transported properly or cleaved.

Structural mapping of cysteine-63 of the chloroplast ATP synthase beta subunit.

The single sulfhydryl residue (cysteine-63) of the beta subunit of the chloroplast ATP synthase F1 (CF1) was accessible to labeling reagents only after removal of the beta subunit from the enzyme complex. This suggests that cysteine-63 may be located at an interface between the beta and the alpha subunits of CF1, although alternative explanations such as a conformational change in beta brought about by its release from CF1 cannot be ruled out. Cysteine-63 was specifically labeled with [(diethylamino)methylcoumarinyl]-maleimide, and the distance between this site and trinitrophenyl-ADP at the nucleotide binding site on beta was mapped using fluorescence resonance energy transfer. Cysteine-63 is located in a hydrophobic pocket, 42 A away from the nucleotide binding site on beta.