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• W2021796789 abstract "The role of subunit a in proton translocation by the Escherichia coliF1Fo ATP synthase is poorly understood. In the membrane-bound Fo sector of the enzyme, H+binding and release occurs at Asp61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp61 via an aqueous access pathway formed at least in part by one or more of the five TMHs of subunit a. In this report, we have substituted Cys into a 19-residue span of the fourth TMH of subunit a and used chemical modification to obtain information about the aqueous accessibility of residues along this helix. Residues 206, 210, and 214 are N-ethylmaleimide-accessible from the cytoplasmic side of the membrane and may lie on the H+ transport route. Residues 215 and 218 on TMH4, as well as residue 245 on TMH5, are Ag+-accessible butN-ethylmaleimide-inaccessible and may form part of an aqueous pocket extending from Asp61 of subunitc to the periplasmic surface. The role of subunit a in proton translocation by the Escherichia coliF1Fo ATP synthase is poorly understood. In the membrane-bound Fo sector of the enzyme, H+binding and release occurs at Asp61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp61 via an aqueous access pathway formed at least in part by one or more of the five TMHs of subunit a. In this report, we have substituted Cys into a 19-residue span of the fourth TMH of subunit a and used chemical modification to obtain information about the aqueous accessibility of residues along this helix. Residues 206, 210, and 214 are N-ethylmaleimide-accessible from the cytoplasmic side of the membrane and may lie on the H+ transport route. Residues 215 and 218 on TMH4, as well as residue 245 on TMH5, are Ag+-accessible butN-ethylmaleimide-inaccessible and may form part of an aqueous pocket extending from Asp61 of subunitc to the periplasmic surface. H+-transporting F1Fo ATP synthases consist of two structurally and functionally distinct sectors termed F1 and Fo (1Senior A.E. Physiol. Rev. 1988; 68: 177-231Google Scholar). In the intact enzyme, ATP synthesis or hydrolysis takes place in the F1 sector and is coupled to active H+ transport through the Fosector. Structurally similar F1Fo ATP synthases are present in mitochondria, chloroplasts, and most eubacteria (1Senior A.E. Physiol. Rev. 1988; 68: 177-231Google Scholar). The F1 sector lies at the surface of the membrane and inEscherichia coli consists of five subunits in an α3β3γ1δ1ε1stoichiometry. The Fo sector spans the membrane and inE. coli consists of three subunits in ana 1 b 2 c 10stoichiometry (2Jiang W. Hermolin J. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4966-4971Google Scholar). The structures of several types of F1have been solved by x-ray crystallography (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Google Scholar, 4Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Google Scholar, 5Menz R.I. Walker J.E. Leslie A.G.W. Cell. 2001; 106: 331-341Google Scholar, 6Shirakihara Y. Leslie A.G.W. Abrahams J.P. Walker J.E. Ueda T. Sekimoto Y. Kambara M. Saika K. Kagawa Y. Yoshida M. Structure. 1997; 5: 825-836Google Scholar, 7Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Google Scholar, 8Groth G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3464-3468Google Scholar). In the case of the bovine mitochondrial enzyme crystallized in the presence of Mg2+ and adenosine phosphates, the three α and three β subunits alternate around the central γ subunit, with subunit γ interacting asymmetrically with the three catalytic sites formed at the αβ interface (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Google Scholar, 4Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Google Scholar, 5Menz R.I. Walker J.E. Leslie A.G.W. Cell. 2001; 106: 331-341Google Scholar). In the widely accepted binding change mechanism for ATP synthesis, the alternate tight binding of ADP and Pi and subsequent release of product ATP are mediated by γ subunit rotation between the alternating catalytic sites (9Boyer P.D. FASEB J. 1989; 3: 2164-2178Google Scholar, 10Leslie A.G. Walker J.E. Philos. Trans. R. Soc. Lond-Biol. Sci. 2000; 355: 465-471Google Scholar, 11Senior A.E. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Google Scholar). Rotation of the γ subunit during ATP hydrolysis was demonstrated by attaching an actin filament to an immobilized α3β3 complex (12Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Google Scholar, 13Kinosita Jr., K. Yasuda R. Noji H. Adachi K. Philos. Trans. R. Soc. Lond-Biol. Sci. 2000; 355: 473-489Google Scholar). In the complete membranous enzyme, the rotation of subunit γ is proposed to be driven by H+ transport-coupled rotation of a connected ring ofc subunits in the Fo sector of the enzyme, which extend through the lipid bilayer and maintain a fixed linkage with the γ subunit. Rotation of the c ring was also demonstrated using the filament rotation assay (14Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Google Scholar, 15Pänke O. Gumbiowski K. Junge W. Engelbrecht S. FEBS Lett. 2000; 472: 34-38Google Scholar). The structure of monomeric subunit c has been solved by NMR in a membrane mimetic solvent mixture (16Girvin M.E. Rastogi V.K. Abildgaard F. Markley J.L. Fillingame R.H. Biochemistry. 1998; 37: 8817-8824Google Scholar), and the structure of the oligomericc 10 ring was predicted from this structure and cross-linking constraints (17Dmitriev O.Y. Jones P.C. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7785-7790Google Scholar, 18Fillingame R.H. Jiang W.P. Dmitriev O.Y. J. Bioenerg. Biomembr. 2000; 32: 433-439Google Scholar). The proposed subunit packing is now supported by a 3.9 Å x-ray diffraction map of an F1 c 10 subcomplex from yeast mitochondria (19Stock D. Leslie A.G.W. Walker J.E. Science. 1999; 286: 1700-1705Google Scholar). The c subunit spans the membrane as a hairpin of two α-helices and in the case of E. colicontains the essential aspartyl 61 residue at the center of the second TMH. 1The abbreviations used are: TMH, transmembrane helix; DTT, dithiothreitol; MTS, methanethiosulfonate; NEM, N-ethylmaleimide; ACMA, 9-amino-6-chloro-2-methoxy acridine; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine 1The abbreviations used are: TMH, transmembrane helix; DTT, dithiothreitol; MTS, methanethiosulfonate; NEM, N-ethylmaleimide; ACMA, 9-amino-6-chloro-2-methoxy acridine; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine Asp61 is thought to undergo protonation and deprotonation as each subunit of the oligomeric ring moves past a stationary subunit a. Subunita is believed to provide access channels to the proton-binding Asp61 residue, but the actual proton translocation pathway remains to be defined (20Fillingame R.H. Jiang W. Dmitriev O.Y. Jones P.C. Biochim. Biophys. Acta. 2000; 1458: 387-403Google Scholar, 21Fillingame R.H. Jiang W. Dmitriev O.Y. J. Exp. Biol. 2000; 203: 9-17Google Scholar, 22Rastogi V.K. Girvin M.E. Nature. 1999; 402: 263-268Google Scholar, 23Cain B.D. J. Bioenerg. Biomembr. 2000; 32: 365-371Google Scholar).The structure of subunit a and its role in H+translocation are poorly defined. Subunit a is known to fold with five TMHs (24Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1998; 273: 16241-16247Google Scholar, 25Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Google Scholar, 26Wada T. Long J.C. Zhang D. Vik S.B. J. Biol. Chem. 1999; 274: 17353-17357Google Scholar) with aTMH4 packing in parallel tocTMH2, i.e. the helix to which Asp61is anchored (27Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Google Scholar). The interaction of the conserved Arg210residue in aTMH4 with cTMH2 is thought to be critical during the deprotonation-protonation cycle ofcAsp61 (23Cain B.D. J. Bioenerg. Biomembr. 2000; 32: 365-371Google Scholar, 28Fillingame R.H. Krulwich T.A. The Bacteria. XII. Academic Press, New York1990: 345-391Google Scholar, 29Hatch L.P. Cox G.B. Howitt S.M. J. Biol. Chem. 1995; 270: 29407-29412Google Scholar, 30Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1997; 272: 32635-32641Google Scholar, 31Wang H.Y. Oster G. Nature. 1998; 396: 279-282Google Scholar, 32Vik S.B. Long J.C. Wada T. Zhang D. Biochim. Biophys. Acta. 2000; 1458: 457-466Google Scholar). The predictedaTMH4/cTMH2 interactions are in accord with second site revertant analysis (33Fraga D. Hermolin J. Fillingame R.H. J. Biol. Chem. 1994; 269: 2562-2567Google Scholar), and cross-link analysis has confirmed closest neighbor proximity of cTMH2 withaTMH4 over a span of 19 amino acid residues (27Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Google Scholar). Both modeling and cross-linking experiments indicate that helix 2 of subunitc should be packed on the outside of the ring (17Dmitriev O.Y. Jones P.C. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7785-7790Google Scholar, 34Jones P.C. Jiang W. Fillingame R.H. J. Biol. Chem. 1998; 273: 17178-17185Google Scholar). Electron microscopic studies support the positioning of subunita and the two b subunits at the periphery of thec ring (35Birkenhager R. Hoppert M. Deckers-Hebestreit G. Mayer F. Altendorf K. Eur. J. Biochem. 1995; 230: 58-67Google Scholar, 36Takeyasu K. Omote H. Nettikadan S. Tokumasu F. Iwamotokihara A. Futai M. FEBS Lett. 1996; 392: 110-113Google Scholar, 37Singh S. Turina P. Bustamante C.J. Keller D.J. Capaldi R. FEBS Lett. 1996; 397: 30-34Google Scholar).The chemical labeling of cysteine side chains introduced by site-directed mutagenesis has been used as a means of mapping aqueous accessible regions on several membrane proteins (38–51). Several reagents have been used to modify the genetically introduced Cys to determine accessibility, including NEM (38Tamura N. Konishi S. Iwaki S. Kimura-Someya T. Nada S. Yamaguchi A. J. Biol. Chem. 2001; 276: 20330-20339Google Scholar, 41Mordoch S.S. Granot D. Lebendiker M. Schuldiner S. J. Biol. Chem. 1999; 274: 19480-19486Google Scholar, 42Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Google Scholar, 43Kwaw I. Zen K.C. Hu Y. Kaback H.R. Biochemistry. 2001; 40: 10491-10499Google Scholar, 44Yan R.T. Maloney P.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5973-5976Google Scholar), MTS reagents (40Wilson G. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1241-1248Google Scholar,48Egan T.M. Haines W.R. Voigt M.M. J. Neurosci. 1998; 18: 2350-2359Google Scholar, 49Rassendren F. Buell G. Newbolt A. North R.A. Surprenant A. EMBO J. 1997; 16: 3446-3454Google Scholar, 50Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Google Scholar, 51Bass R.B. Coleman M.D. Falke J.J. Biochemistry. 1999; 38: 9317-9327Google Scholar), and Ag+ (45Lu Q. Miller C. Science. 1995; 268: 304-307Google Scholar, 46Kriegler S. Sudweeks S. Yakel J.L. J. Biol. Chem. 1999; 274: 3934-3936Google Scholar, 47del Camino D. Yellen G. Neuron. 2001; 32: 649-656Google Scholar, 48Egan T.M. Haines W.R. Voigt M.M. J. Neurosci. 1998; 18: 2350-2359Google Scholar). Modification of Cys by these reagents depends upon ionization of the Cys sulfhydryl to its thiolate form (52Gorin G. Martic P.A. Doughty G. Arch. Biochem. Biophys. 1966; 115: 593-597Google Scholar, 53Friedman M. The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins. 1st Ed. Pergamon Press, Oxford1973Google Scholar, 54Roberts D.D. Lewis S.D. Ballou D.P. Olson S.T. Shafer J.A. Biochemistry. 1986; 25: 5595-5601Google Scholar, 55Dance I.G. Polyhedron. 1986; 5: 1037Google Scholar), and this is expected to occur preferentially in an aqueous environment (38Tamura N. Konishi S. Iwaki S. Kimura-Someya T. Nada S. Yamaguchi A. J. Biol. Chem. 2001; 276: 20330-20339Google Scholar, 39Li J. Xu Q. Cortes D.M. Perozo E. Laskey A. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11605-11610Google Scholar, 41Mordoch S.S. Granot D. Lebendiker M. Schuldiner S. J. Biol. Chem. 1999; 274: 19480-19486Google Scholar). In this report, a span of 19 residues in aTMH4 were replaced with Cys and tested for accessibility to water-soluble reagents NEM and Ag+. We found that residues 206, 210, and 214 are NEM-accessible from the cytoplasmic side of the membrane. In contrast, residues 215 and 218 on helix 4 and residue 245 on helix 5 form an Ag+-accessible but NEM-inaccessible pocket bridging aTMH4 and aTMH5. This pocket may form part of the aqueous access pathway extending from Asp61 to the periplasmic surface.DISCUSSIONIn this paper, we report on the aqueous accessibility of cysteine residues substituted into the fourth transmembrane helix in subunita of the rotary ATP synthase. aTMH4 is thought to interact with Asp61 of subunit c during proton translocation and may play a part in forming an aqueous access channel to cAsp61 from one or both sides of the membrane. The proximity of aTMH4 to cTMH2 was initially postulated on the basis of second site suppressor analysis (33Fraga D. Hermolin J. Fillingame R.H. J. Biol. Chem. 1994; 269: 2562-2567Google Scholar) and is now supported by intermolecular Cys-Cys cross-links betweencTMH2 and aTMH4 (27Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Google Scholar). Aqueous access to Asp61 of subunit c had been postulated previously based upon the pH-sensitive function of the cA24D mutant (67Zhang Y. Fillingame R.H. J. Biol. Chem. 1994; 269: 5473-5479Google Scholar) and is further supported by the discovery that simultaneous mutation of three residues surrounding Asp61 makes the enzyme sensitive to inhibition by Li+ (68Zhang Y. Fillingame R.H. J. Biol. Chem. 1995; 270: 87-93Google Scholar). Additionally, the analogous enzyme from Propiogenium modestum, with a very homologous subunit c, alternatively transports Na+, Li+, or H+ (69Dimroth P. Biochim. Biophys. Acta. 2000; 1458: 374-386Google Scholar, 70Kaim G. Biochim. Biophys. Acta. 2001; 1505: 94-107Google Scholar). Thus, it seems likely that these various ions gain access to the membrane-embedded carboxyl of subunit c via a water-filled channel. We have substituted cysteine over the length ofaTMH4 and tested the susceptibility of each substitution to modification with water-soluble, thiol-modifying reagents. The approach has been used previously to define surfaces of membrane proteins with aqueous accessibility (38–51).Reactivity of Cys Substitutions with NEM, Ag+, and MTS ReagentsThe ionized sulfhydryl group of cysteine is the form that preferentially reacts with the thiol-specific reagents used here. For example, the reactivity of MTS reagents with the thiolate is preferred by a factor of 109 over reaction with a nonionized thiol group (54Roberts D.D. Lewis S.D. Ballou D.P. Olson S.T. Shafer J.A. Biochemistry. 1986; 25: 5595-5601Google Scholar). Similarly, NEM (52Gorin G. Martic P.A. Doughty G. Arch. Biochem. Biophys. 1966; 115: 593-597Google Scholar, 53Friedman M. The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins. 1st Ed. Pergamon Press, Oxford1973Google Scholar) and Ag+ (45Lu Q. Miller C. Science. 1995; 268: 304-307Google Scholar, 55Dance I.G. Polyhedron. 1986; 5: 1037Google Scholar) react preferentially with ionized thiolates rather than with neutral thiols. The differential reactivity of substituted cysteines can thus provide information about the ionization state of different residues, which in most cases should be related to aqueous accessibility, i.e. the interpretation given in similar studies of other membrane proteins (38Tamura N. Konishi S. Iwaki S. Kimura-Someya T. Nada S. Yamaguchi A. J. Biol. Chem. 2001; 276: 20330-20339Google Scholar, 39Li J. Xu Q. Cortes D.M. Perozo E. Laskey A. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11605-11610Google Scholar, 41Mordoch S.S. Granot D. Lebendiker M. Schuldiner S. J. Biol. Chem. 1999; 274: 19480-19486Google Scholar). The means by which NEM penetrates the membrane to react with Cys residues is not certain. It may be sufficiently lipid-soluble to gain access to transmembrane Cys via the hydrophobic phase of the membrane. However, modification should only be observed with those residues subject to ionization. It is also conceivable that uncharged MTS reagents could access reactive Cys residues via the lipid phase, although reactivity would again depend upon the ionization state of the sulfhydryl group. It is of interest that Ag+ reacts with transmembrane residues that are NEM-insensitive. This may indicate that NEM-sensitive residues need to be bounded by larger aqueous cavities, sufficient in size to accommodate the bulkier NEM molecule.NEM Reactive TMH4 SubstitutionsResidues 206, 210, and 214 onaTMH4 are preferentially modified with NEM, as indicated by direct labeling with [14C]NEM and also by inhibition of ATP-driven quenching in the case of the 206 and 214 mutants. The radioactive labeling studies were carried out to address the possibility that residues might be modified with NEM without effect on function. Additionally, we were able to examine the labeling of a Cys at position 210 in a mutant that is nonfunctional in proton transport. These three residues fall on one face of an α-helix (Fig.13), and it seems likely that NEM modification results in a block to proton transport through this region of the protein. Inhibition of proton translocation by attachment of the ethylmaleimide group is consistent with evidence from mutagenesis studies that bulky substitutions are not as well tolerated as smaller ones at these positions (Table III). The fact that the three residues found to label with NEM line one face ofaTMH4 suggests that NEM could be gaining access to these residues via an aqueous pathway formed at this face of an α-helix.Table IIIProperties of aTMH4 mutations constructed previously in NEM or Ag+-sensitive residuesPositionSubstitutionGrowth on SuccinateaIn most cases ATP-driven proton pumping was also measured, and the decrease in activity correlates with the growth on succinate parameter.ReferenceSer206Ala+++Ref.73Howitt S.M. Gibson F. Cox G.B. Biochim. Biophys. Acta. 1988; 936: 74-80Google ScholarLeuSlowRef. 74Cain B.D. Simoni R.D. J. Biol. Chem. 1986; 261: 10043-10050Google ScholarArg210Ala0Ref. 29Hatch L.P. Cox G.B. Howitt S.M. J. Biol. Chem. 1995; 270: 29407-29412Google ScholarVal0Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarIle0Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarGln0Ref. 29Hatch L.P. Cox G.B. Howitt S.M. J. Biol. Chem. 1995; 270: 29407-29412Google ScholarGlu0Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarLys0Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarGly213Asp0Unpublished dataAsn214Val+++Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarLeu+Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarGlu+++Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarGln+++Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarHis0Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarGly218Ala+++Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarVal+Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarAsp+Refs. 66Hartzog P.E. Cain B.D. J. Biol. Chem. 1994; 269: 32313-32317Google Scholar and 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google ScholarLys−Ref. 75Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Google Scholara In most cases ATP-driven proton pumping was also measured, and the decrease in activity correlates with the growth on succinate parameter. Open table in a new tab The Cys206 residue appears to titrate as the pH of the medium is lowered from 7.5 to 7.0, as indicated by the dramatic decrease in NEM reactivity, suggesting that this residue is accessible to the bulk solvent. The solvent accessibility of this residue is also supported by the rapid reversal of Ag+ inhibition by dithiothreitol. On the other hand, the reactivity of Cys214is unaffected by lowering the pH of the medium to 7.0, suggesting that this residue remains ionized at a lower pH than Cys206, even though topological analysis would place residue 214 in the middle of the membrane. Despite its low apparent pK a, Cys214 does not appear to be generally accessible to the aqueous medium based upon the slow reversal of Ag+inhibition by DTT. We suggest that the low pK a of Cys214 at the center of the membrane may be due to salt bridge formation with the proximal Arg210 residue.Silver Used to Probe aTMH4 Cys SubstitutionsSilver has been used previously as an irreversible covalent modifier of Cys introduced into several membrane proteins (45Lu Q. Miller C. Science. 1995; 268: 304-307Google Scholar, 46Kriegler S. Sudweeks S. Yakel J.L. J. Biol. Chem. 1999; 274: 3934-3936Google Scholar, 47del Camino D. Yellen G. Neuron. 2001; 32: 649-656Google Scholar, 48Egan T.M. Haines W.R. Voigt M.M. J. Neurosci. 1998; 18: 2350-2359Google Scholar). The irreversibility of the Ag+ inhibition under the conditions used here is indicated by an experiment where Ag+ pretreatment of membrane vesicles protected Cys215 from becoming labeled with [14C]NEM after solubilization of the membrane with SDS (experiment not shown). Further, if membranes are treated with silver and centrifuged to remove the assay buffer, inhibition is still observed on resuspension in Ag+-free buffer with Cl− present to precipitate any free silver released to solution. As would be expected (65Bell R.A. Kramer J.R. Environ. Toxicol. Chem. 1999; 18: 9-22Google Scholar), the reaction and inhibition can in some cases be reversed by competing sulfhydryl reagents such as DTT,e.g. in the case of the aS206C substitution. It is not clear from our studies exactly how silver gains access to these sulfhydryls. In this experimental system, we have shown by atomic absorption analysis that 80% of silver added to membrane vesicles becomes complexed with the membrane. Conceivably, the silver may be transported to the interior of the membrane vesicles, where it gains access to subunit a. Alternatively, the silver may bind to other membrane proteins or sites of unsaturation on fatty acid tails (71Bennett M.A. Chem. Rev. 1962; 62: 611-652Google Scholar, 72Morris L.J. J. Lipid Res. 1966; 7: 717-732Google Scholar). To test whether other proteins in the membrane were transporting silver or were necessary for silver absorption by the membrane, F1Fo was purified and reconstituted into liposomes and tested for Ag+ inhibition of ATP-driven proton pumping by Ag+. The Cys214 and Cys215 mutant enzymes still exhibited inhibition by Ag+, suggesting that the silver may gain access to these cysteines without the aid of other membrane proteins and that the inhibitory Ag+ does not need to be transported to the interior of these vesicles by an inner membrane Ag+transport system.As seen in Fig. 12, aH245C/D119H shows inhibition of ATP-driven quenching when treated with silver. This is the first direct evidence that the aqueous access pathway tocAsp61 may involve more than one helix of subunit a. Looking at the silver-sensitive residues highlighted on the model of subunit a in Fig. 13, it is certainly possible that these residues form a pocket that is accessed from an aqueous channel extending to the cytoplasmic side of the membrane. The aqueous pathway to the cytoplasm appears to include NEM-sensitive residues 206, 210, and 214 on one α-helical face ofaTMH4. To the periplasmic side ofaAsn214, residues 215–220 are characterized here as being NEM-inaccessible but Ag+-sensitive, with residues 215 and 218 showing the greatest silver sensitivity. Residues 215 and 218 would fall on the α-helical face of aTMH4 opposite to residues 206, 210, and 214. If an aqueous pocket bridges these residues and residue 245 in aTMH5, thenaTMH4 may have to swivel to gate access between the periplasmic pocket and Asp61 of subunit c. As we have discussed elsewhere (76Fillingame R.H. Angevine C.M. Dmitriev O.Y. Biochim. Biophys. Acta. 2002; 1555: 29-36Google Scholar), such swiveling may be coupled mechanically to other helical movements that drive stepwise rotation of the c ring. H+-transporting F1Fo ATP synthases consist of two structurally and functionally distinct sectors termed F1 and Fo (1Senior A.E. Physiol. Rev. 1988; 68: 177-231Google Scholar). In the intact enzyme, ATP synthesis or hydrolysis takes place in the F1 sector and is coupled to active H+ transport through the Fosector. Structurally similar F1Fo ATP synthases are present in mitochondria, chloroplasts, and most eubacteria (1Senior A.E. Physiol. Rev. 1988; 68: 177-231Google Scholar). The F1 sector lies at the surface of the membrane and inEscherichia coli consists of five subunits in an α3β3γ1δ1ε1stoichiometry. The Fo sector spans the membrane and inE. coli consists of three subunits in ana 1 b 2 c 10stoichiometry (2Jiang W. Hermolin J. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4966-4971Google Scholar). The structures of several types of F1have been solved by x-ray crystallography (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Google Scholar, 4Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Google Scholar, 5Menz R.I. Walker J.E. Leslie A.G.W. Cell. 2001; 106: 331-341Google Scholar, 6Shirakihara Y. Leslie A.G.W. Abrahams J.P. Walker J.E. Ueda T. Sekimoto Y. Kambara M. Saika K. Kagawa Y. Yoshida M. Structure. 1997; 5: 825-836Google Scholar, 7Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Google Scholar, 8Groth G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3464-3468Google Scholar). In the case of the bovine mitochondrial enzyme crystallized in the presence of Mg2+ and adenosine phosphates, the three α and three β subunits alternate around the central γ subunit, with subunit γ interacting asymmetrically with the three catalytic sites formed at the αβ interface (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Google Scholar, 4Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Google Scholar, 5Menz R.I. Walker J.E. Leslie A.G.W. Cell. 2001; 106: 331-341Google Scholar). In the widely accepted binding change mechanism for ATP synthesis, the alternate tight binding of ADP and Pi and subsequent release of product ATP are mediated by γ subunit rotation between the alternating catalytic sites (9Boyer P.D. FASEB J. 1989; 3: 2164-2178Google Scholar, 10Leslie A.G. Walker J.E. Philos. Trans. R. Soc. Lond-Biol. Sci. 2000; 355: 465-471Google Scholar, 11Senior A.E. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Google Scholar). Rotation of the γ subunit during ATP hydrolysis was demonstrated by attaching an actin filament to an immobilized α3β3 complex (12Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Google Scholar, 13Kinosita Jr., K. Yasuda R. Noji H. Adachi K. Philos. Trans. R. Soc. Lond-Biol. Sci. 2000; 355: 473-489Google Scholar). In the complete membranous enzyme, the rotation of subunit γ is proposed to be driven by H+ transport-coupled rotation of a connected ring ofc subunits in the Fo sector of the enzyme, which extend through the lipid bilayer and maintain a fixed linkage with the γ subunit. Rotation of the c ring was also demonstrated using the filament rotation assay (14Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Google Scholar, 15Pänke O. Gumbiowski K. Junge W. Engelbrecht S. FEBS Lett. 2000; 472: 34-38Google Scholar). The structure of monomeric subunit c has been solved by NMR in a membrane mimetic solvent mixture (16Girvin M.E. Rastogi V.K. Abildgaard F. Markley J.L. Fillingame R.H. Biochemistry. 1998; 37: 8817-8824Google Scholar), and the structure of the oligomericc 10 ring was predicted from this structure and cross-linking constraints (17Dmitriev O.Y. Jones P.C. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7785-7790Google Scholar, 18Fillingame R.H. Jiang W.P. Dmitriev O.Y. J. Bioenerg. Biomembr. 2000; 32: 433-439Google Scholar). The proposed subunit packing is now supported by a 3.9 Å x-ray diffraction map of an F1 c 10 subcomplex from yeast mitochondria (19Stock D. Leslie A.G.W. Walker J.E. Science. 1999; 286: 1700-1705Google Scholar). The c subunit spans the membrane as a hairpin of two α-helices and in the case of E. colicontains the essential aspartyl 61 residue at the center of the second TMH. 1The abbreviations used are: TMH, transmembrane helix; DTT, dithiothreitol; MTS, methanethiosulfonate; NEM, N-ethylmaleimide; ACMA, 9-amino-6-chloro-2-methoxy acridine; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine 1The abbreviations used are: TMH, transmembrane helix; DTT, dithiothreitol; MTS, methanethiosulfonate; NEM, N-ethylmaleimide; ACMA, 9-amino-6-chloro-2-methoxy acridine; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine Asp61 is thought to undergo protonation and deprotonation as each subunit of the oligomeric ring moves past a stationary subunit a. Subunita is believed to provide access channels to the proton-binding Asp61 residue, but the actual proton translocation pathway remains to be defined (20Fillingame R.H. Jiang W. Dmitriev O.Y. Jones P.C. Biochim. Biophys. Acta. 2000; 1458: 387-403Google Scholar, 21Fillingame R.H. Jiang W. Dmitriev O.Y." @default.
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• W2021796789 title "Aqueous Access Channels in Subunit a of Rotary ATP Synthase" @default.
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