Rotational Catalysis of Escherichia coli ATP Synthase F1 Sector
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m700551200
ISSN1083-351X
AutoresMayumi Nakanishi‐Matsui, Sachiko Kashiwagi, Toshiharu Ubukata, Atsuko Iwamoto-Kihara, Yoh Wada, Masamitsu Futai,
Tópico(s)RNA modifications and cancer
ResumoA complex of γ, ∊, and c subunits rotates in ATP synthase (FoF1) coupled with proton transport. A gold bead connected to the γ subunit of the Escherichia coli F1 sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the β subunit key region; consistent with its bulk phase ATPase activities, F1 with the Ser-174 to Phe substitution (βS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120° positions during the stepped revolution. The pause positions were probably not at the “ATP waiting” dwell but at the “ATP hydrolysis/product release” dwell, since the ATP concentration used for the assay was ∼30-fold higher than the Km value for ATP. A βGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of βS174F. The revertant (βG149A/βS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant βG149A/βS174L. These results indicate that the domain between β-sheet 4 (βSer-174) and P-loop (βGly-149) is important to drive rotation. A complex of γ, ∊, and c subunits rotates in ATP synthase (FoF1) coupled with proton transport. A gold bead connected to the γ subunit of the Escherichia coli F1 sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the β subunit key region; consistent with its bulk phase ATPase activities, F1 with the Ser-174 to Phe substitution (βS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120° positions during the stepped revolution. The pause positions were probably not at the “ATP waiting” dwell but at the “ATP hydrolysis/product release” dwell, since the ATP concentration used for the assay was ∼30-fold higher than the Km value for ATP. A βGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of βS174F. The revertant (βG149A/βS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant βG149A/βS174L. These results indicate that the domain between β-sheet 4 (βSer-174) and P-loop (βGly-149) is important to drive rotation. A ubiquitous ATP synthase (FoF1) synthesizes ATP coupled with an electrochemical proton gradient formed by a respiratory chain (for reviews, see Refs 1Futai M. Sun-Wada G.H. Wada Y. Futai M. Wada Y. Kaplan J. Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany2004: 237-260Google Scholar, 2Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1576) Google Scholar, 3Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar, 4Stock D. Gibbons C. Arechaga I. Leslie A.G.W. Walker J.E. Curr. Opin. Sruct. Biol. 2000; 10: 672-679Crossref PubMed Scopus (260) Google Scholar, 5Fillingame R.H. Angevine C.M. Dmitriev O.Y. FEBS Lett. 2003; 555: 29-34Crossref PubMed Scopus (128) Google Scholar). 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Muneyuki E. Hara K.Y. Kinosita Jr., K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14731-14736Crossref PubMed Scopus (214) Google Scholar, 22Nishizaka T. Oiwa K. Noji H. Kimura S. Muneyuki E. Yoshida M. Kinosita Jr., K. Nat. Struct. Mol. Biol. 2004; 11: 142-148Crossref PubMed Scopus (240) Google Scholar). We have observed that the rotation speed of beads attached to the Escherichia coli γ subunit varied, reflecting stochastic fluctuations (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 23Nakanishi-Matsui M. Futai M. IUBMB Life. 2006; 58: 318-322Crossref PubMed Scopus (23) Google Scholar). Although the average speeds were dependent on the diameter of beads, 40- and 60-nm diameter beads rotated with essentially the same rate (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), suggesting that their rotation speeds were close to that of the γ subunit without a probe attached. The mechanism underlying the chemistry and energy coupling of FoF1 has been studied by introducing mutations (1Futai M. Sun-Wada G.H. Wada Y. Futai M. Wada Y. Kaplan J. Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany2004: 237-260Google Scholar, 10Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (124) Google Scholar, 13Tanabe M. Nishio K. Iko Y. Sambongi Y. Iwamoto-Kihara A. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 15269-15274Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Hosokawa H. Nakanishi-Matsui M. Kashiwagi S. Fujii-Taira I. Hayashi K. Iwamoto-Kihara A. Wada Y. Futai M. J. Biol. Chem. 2005; 280: 23797-23801Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 24Iko Y. Sambongi Y. Tanabe M. Iwamoto-Kihara A. Saito K. Ueda I. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 47508-47511Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). One of the most interesting mutations is the substitution of the βSer-174 residue (24Iko Y. Sambongi Y. Tanabe M. Iwamoto-Kihara A. Saito K. Ueda I. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 47508-47511Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 25Omote H. Park M-Y. Maeda M. Futai M. J. Biol. Chem. 1994; 269: 10265-10269Abstract Full Text PDF PubMed Google Scholar, 26Iwamoto A. Omote H. Hanada H. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 16350-16355Abstract Full Text PDF PubMed Google Scholar, 27Iwamoto A. Park M-Y. Maeda M. Futai M. J. Biol. Chem. 1993; 268: 3156-3160Abstract Full Text PDF PubMed Google Scholar) located in β-sheet 4, thus being distant from the bound ATP in the β subunit (8Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2743) Google Scholar) (see Fig. 1). The size of the residue at this position is pertinent as to the activity (25Omote H. Park M-Y. Maeda M. Futai M. J. Biol. Chem. 1994; 269: 10265-10269Abstract Full Text PDF PubMed Google Scholar); the larger the side-chain volume of the residue introduced, the lower the ATPase activity became. The βS174F (βSer-174 to Phe substitution) or βS174L (βSer-174 to Leu) F1 sector exhibited ∼10% of the wild-type activity (25Omote H. Park M-Y. Maeda M. Futai M. J. Biol. Chem. 1994; 269: 10265-10269Abstract Full Text PDF PubMed Google Scholar). The defect of βS174F was suppressed by the replacement of βGly-149 (βGly-149 to Ala, Ser, or Cys) in the phosphate binding P-loop near α-helix B (26Iwamoto A. Omote H. Hanada H. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 16350-16355Abstract Full Text PDF PubMed Google Scholar, 27Iwamoto A. Park M-Y. Maeda M. Futai M. J. Biol. Chem. 1993; 268: 3156-3160Abstract Full Text PDF PubMed Google Scholar). However, rotation of an actin filament connected to γ was not proportional to the ATPase activity; generated torque for βS174F and βS174L were 40∼100% that of the wild-type level (24Iko Y. Sambongi Y. Tanabe M. Iwamoto-Kihara A. Saito K. Ueda I. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 47508-47511Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Thus, it became of interest to analyze mechanical revolution of the γ subunit in these mutants using a probe smaller than an actin filament. In this study we confirmed stochastic fluctuation of the γ subunit rotation by analyzing the single revolution time (time required for 360° revolution). The βS174F mutant paused at 120° steps longer than the wild type, giving an ∼6 times lower revolution time. The rotation of second-site revertant βG149A/βS174F was similar to that of the wild type. Essentially the same results were obtained for βS174L mutant and its second-site revertant βG149A/βS174L. We discuss a possible role(s) of the domain including β-sheet 4 and the phosphate binding P-loop, where βSer-174 and βGly-149 are located, respectively (Fig. 1). Preparation and Materials—E. coli strain DK8 (ΔuncB-C) was used as a host for recombinant plasmids and grown at 37 °C in a synthetic medium containing 0.5% glycerol as a carbon source. A plasmid carrying the unc operon introduced six His residues at the α subunit amino terminus, and γS193C and γK108C substitutions in the γ subunit were described previously (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Mutations βS174F and βS174L and related substitutions of the β subunit were introduced into F1 engineered for rotation (24Iko Y. Sambongi Y. Tanabe M. Iwamoto-Kihara A. Saito K. Ueda I. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 47508-47511Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The wild-type and mutant F1 sectors were purified on a glycerol gradient (24Iko Y. Sambongi Y. Tanabe M. Iwamoto-Kihara A. Saito K. Ueda I. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 47508-47511Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Gel electrophoresis showed the presence of about 1 mol ∊/mol F1 but no δ subunit in any of the preparations analyzed. Gold beads (60-nm diameter) were obtained from British Bio Cell International and were coated with biotinylated bovine serum albumin (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The cover glass used for constructing a cell for observing rotation was soaked in 0.1 n KOH for 3 days followed by extensive washing with high purity water (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Assay Procedures—Mutant and wild-type ATPase activities were assayed as described previously (10Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (124) Google Scholar) under the conditions used for the rotation assay (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The third (highest) Km values for ATP were obtained with the Mg2+ concentrations that gave the maximal activities. When varying the ATP concentration, the Mg2+:ATP ratio was maintained at 1:1, except that the Mg2+ concentration was 0.5 mm when ATP concentration was lower than 0.5 mm. For the βG149A mutant, the ratio was maintained at 2:1, except that the Mg2+ concentration was 4 mm for ATP lower than 0.5 mm. Protein concentrations were determined using bovine serum albumin (Sigma, Fraction V) as a standard (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). Observing γ Subunit Rotation—Rotation was assayed essentially as described previously (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Briefly, gold beads were connected to the immobilized F1 sector in a flow cell (∼30-μm deep) filled with buffer A (10 mg/ml bovine serum albumin, 10 mm MOPS 2The abbreviation used is: MOPS, 3-(N-morpholino) propanesulfonic acid. /KOH, pH 7.0, 50 mm KCl, and 2 mm MgCl2). Immediately after the introduction of buffer A containing 2 mm ATP and its regenerating system, images of the beads illuminated with laser light (JUNO EX, Showa Optronics Co.) were obtained on dark field microscopy (BX51WI-CDEVA-F, Olympus, Tokyo) and recorded with a charge-coupled device camera for data analysis using a Metamorph (Molecular Devices Corp.). The proper camera speeds (1000∼4000 frames/s) were selected depending on the mutations of the F1 sectors; wild-type and the revertants, 4000 frames/s; βS174F, βS174L, and βG149A, 1000 frames/s. They were also assayed at a different camera speed when necessary. Other methods, including construction of glass cells for rotation and laser-light illumination, were described previously (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We occasionally observed apparent clockwise movements of less than two revolutions, although they were not actual rotations. The beads analyzed were those showing such movements amounting to less than 5% of the total counterclockwise revolutions. Stochastic Rotation of Gold Beads Attached to Wild-type F1—Gold beads connected to F1 rotated with various speeds and often paused for a short period (∼ms) as shown previously (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In the previous paper, we recorded their rotations for 0.25 s, estimated rates every 10 ms, and combined those from different beads (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Histograms of the rotation rates showed stochastic fluctuation. However, previous analysis may emphasize the frequencies of rates close to 0 rps if beads paused a long time (>10 ms). Furthermore, these histograms may include variation between beads together with fluctuation of the rotation speeds. The previous observation time (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) was not enough to detect long pauses (>0.25 s). Thus, we were prompted to study longer time courses and single rotation events (360° rotations). The present analysis was more appropriate than estimating rates every 10 ms (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), because all revolutions could be included. In this study the time course of each bead was followed for 2 s. We occasionally observed long pauses (>0.1 s) (Fig. 2a), which were not found in previous time courses (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar); a bead rotated about 500 revolutions and paused (yellow curve in Fig. 2a), and others paused after 300 revolutions (green and pink curves). Sometimes we observed beads that started rotating after a long pause (gray curve). On average, about 3 long pauses appeared when we recorded 1000 revolutions. These pauses were possibly because of Mg-ADP inhibition (29Hirono-Hara Y. Noji H. Nishiura M. Muneyuki E. Hara K.Y. Yasuda R. Kinosita Jr., K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13649-13654Crossref PubMed Scopus (159) Google Scholar), as discussed below. We assumed that the long dwells are Mg-inhibited states of the enzyme and analyzed single rotation events. When time courses were expanded, apparent smooth rotations (Fig. 2a) exhibited dwells (short pauses, ∼ms) and ∼120° stepping (Fig. 2b). Therefore, we analyzed the single revolution time, i.e. the time required for 360° of revolution, to evaluate stochastic fluctuation of rotation. This parameter could follow all rotations of a bead in a time course regardless of the length of the pauses. As expected from the various speeds observed in a time course, each bead clearly showed stochastic fluctuation of the single revolution time (for examples, see Fig. 2c). The histograms for the individual beads and those for multiple beads (Fig. 2d) are closely similar, indicating that fluctuation tendency is an intrinsic property of the F1 molecule. The geometric mean of single revolution time was ∼2.3 ms (2.0, 2.3, 2.3, and 2.6 ms, for four different beads) (Fig. 2c), and the average rotation rate (reciprocal of single revolution time) was 440 rps, i.e. slightly higher than the reported value, 380 rps (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A 360° rotation including the long pause was observed with lower frequency and shown in the histograms of single rotation times (see >15, horizontal axis in Fig. 2, c and d). ATPase Activity of Mutant F1—It became of interest to analyze the mutant F1 sector to understand the mechanisms underlying rotation and its stochastic fluctuation. βS174F (Fig. 1b) and related mutations were introduced into the engineered F1 to observe rotation as previously described (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 24Iko Y. Sambongi Y. Tanabe M. Iwamoto-Kihara A. Saito K. Ueda I. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 47508-47511Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Steady state ATPase activities were assayed under the same conditions as those for rotation observation. The relative activities (% of wild-type level) were essentially similar to previous results: βS174F, 8%; βS174L, 9%; βG149A/βS174F, 314%; βG149A/βS174L, 200%; βG149A, 75%. We also estimated the third (highest) Km for ATP to assay rotation in the presence of excess ATP. βS174F, βS174L, and the second-site revertant βG149A/βS174F exhibited Km values for ATP of 69, 60, and 64 μm, respectively, i.e. essentially the same as that of the wild type (71 μm). βG149A exhibited a Km value (0.3 mm) of ∼4-fold higher than that of the wild type, possibly because of the location of βGly-149 near bound-Mg-ATP at the catalytic site (Fig. 1c). Based on these biochemical properties of the mutant enzymes, we assayed rotations of βS174F, βS174L, and the corresponding second-site revertants in the presence of 2 mm ATP, ∼30-fold higher than the Km values to compare steady state ATP hydrolysis and rotation. Rotation of Mutant F1 with βSer-174 Replacement—The time course of a gold bead attached to βS174F was followed for 2 s. The mutant rotated with variable rates and exhibited long pauses similar to the wild type (Fig. 3, a and b). The total revolutions (in 2 s) were much less than those of the wild type (Fig. 3b). Thus, we compared the wild type and mutants by analyzing single rotation events. The mutant apparently exhibited longer single revolution times than the wild type, as shown for the histograms of multiple beads (Fig. 3c). They are similar to those for single beads (data not shown). The peaks of the wild-type and mutant histograms from multiple beads were at 1.75 and 5 ms, respectively, and their geometric means were 2.3 and 14 ms, respectively. Most (∼80%) of the mutant single revolution times were longer than 5 ms, and ∼50% of them were longer than 10 ms (Fig. 3c), whereas ≥90% of those of the wild type were ≤5 ms. The geometric mean of the βS174L single revolution times was ∼26 ms, i.e. ∼11 times longer time than that of the wild type, and >20% of the mutant times were longer than 100 ms, as shown by the histograms (Fig. 3d). These results were consistent with the low ATPase activities of the mutants. Pausing of the γ Subunit in the Presence of a High Concentration of ATP—The single revolution times of βS174F and βS174L were significantly longer than those of the wild type in the presence of 2 mm ATP (Fig. 3, c and d), possibly because the mutants exhibited a high tendency for longer dwell. Thus, we compared dwells of the wild type and mutants. As shown by expanded time courses, the mutant paused longer at 1/3, 2/3, or 3/3 of a 360° revolution compared with the wild type (Fig. 4a, note the time scale). The angular distributions of the centroids showed three peaks at about 120°, 240°, and 360°/0° (Fig. 4b), indicating that beads paused after 120° revolutions, as shown previously with low ATP concentrations (20Yasuda R. Noji H. Kinosita Jr., K. Yoshida M. Cell. 1998; 93: 1117-1124Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar). However, the pausing observed in the present study may not be at the ATP waiting dwell (time for a catalytic site waiting for ATP binding), since it was not affected by a further increase in the ATP concentration (data not shown), and rotations were assayed in the presence of a ∼30-fold higher ATP concentration than the Km. The pausing dwell contributed significantly to a single revolution time since the stepping (0 → 120°, 120 → 240°, or 240° → 360°/0°) speeds were high (mostly ≤0.25 and ≤1 ms per 120° step for wild-type and mutant, respectively). About 80% of the pausing dwells of the wild type and mutant were ≤0.5 and ≤5ms, respectively (Fig. 4c), indicating that mutant paused at least ∼10 times longer than the wild type. Essentially the same results were obtained for the βS174L mutant. Thus, the longer pausing dwells caused the slow rotation speeds and low ATPase activities of mutants. Rotation of the Second-site Revertant—As shown previously, a defect of the βS174F enzyme was suppressed by the second-site mutations, βGly-149 to Ser, Cys, or Ala (26Iwamoto A. Omote H. Hanada H. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 16350-16355Abstract Full Text PDF PubMed Google Scholar, 27Iwamoto A. Park M-Y. Maeda M. Futai M. J. Biol. Chem. 1993; 268: 3156-3160Abstract Full Text PDF PubMed Google Scholar), giving apparently wild-type ATPase activity. The time course of the revertant βG149A/βS174F and βG149A single mutant was analyzed (Fig. 5, a and b) and shown together with those of the wild type and βS174F mutant for comparison (Fig. 5c). Beads attached to the revertant F1 rotated similar to those of the wild type, except that they rarely exhibited pauses longer than 0.1 s. The histogram of the single revolution times for the revertant showed a peak at 2.5 ms and a geometric mean of 2.9 ms (Fig. 5d), i.e. similar to the wild-type values (Fig. 2d). The average rotation rates were estimated as reciprocals of the geometric means for single revolution times, assuming that F1 sectors exhibiting various speeds were present in the assay mixture; those for the wild type, βS174F, revertant, and βG149A were 440, 70, 340, and 70 rps, respectively. The results for the two single mutants and the revertant indicate that the two mutations (βS174F and βG149A) suppressed each other and gave similar rates as the wild type. Essentially the same results were obtained for βG149A/βS174L (Fig. 5e), the second-site revertant of βS174L. We have previously observed that a gold bead attached to the γ subunit rotated at various rates, indicating stochastic fluctuation of F1 rotation (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, the reported results may have included variations between F1 molecules because data for multiple beads were combined to obtain histograms. In this study we analyzed single revolution times that include the pausing dwell and stepping velocity. As expected from the time courses, individual beads exhibited stochastic fluctuations, and their histograms were similar, indicating that the variation among F1 molecule was not significant. The observed stochastic fluctuation was probably because of the intrinsic properties of the γ subunit driven by catalysis in α3β3 hexamer, since they were essentially independent of the bead sizes (40-200-nm diameter) (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 23Nakanishi-Matsui M. Futai M. IUBMB Life. 2006; 58: 318-322Crossref PubMed Scopus (23) Google Scholar), lengths of histidine tag (6 or 10 histidine residues) introduced into the α subunit, 3M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, and M. Futai, unpublished observation. and enzyme preparations (F1 or FoF1) (data not shown). The fluctuations were mainly because of the varying pausing dwells (∼ms) because stepping was fast. Stochastic fluctuation was also observed in Bacillus F1 with the fluorophore Cy3, attached to the γ subunit (30Adachi K. Yasuda R. Noji H. Itoh H. Harada Y. Yoshida M. Kinosita Jr., K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7243-7247Crossref PubMed Scopus (185) Google Scholar). Careful definition of wild-type rotation was the basis of further studies on mutant F1. Replacement of βSer-174 in β-sheet 4 lowered the ATPase activity to ∼10% of the wild-type level (25Omote H. Park M-Y. Maeda M. Futai M. J. Biol. Chem. 1994; 269: 10265-10269Abstract Full Text PDF PubMed Google Scholar). The means of single revolution times of the beads attached to βS174F and βS174L were about 6 and 11 times longer than that of the wild type, respectively, consistent with about a 10 times longer pausing dwell of the mutant than that of the wild type. βS174L took longer single revolution times than βS174F for an unknown reason, although their steady state ATPase activities were similar (9 and 8% of the wild-type level, respectively). The difference between the results of ATPase activity and single revolution time may be because of the observation times: rotation assay, 2 s; ATPase activity, 3 min. It is possible that βS174F did not rotate for long time (>2s) more often than βS174L. βS174F and βS174L exhibited the ∊ subunit sensitivity similar to the wild type (data not shown), suggesting that the slow rotations by the mutants were not because of the effect of this subunit. As shown previously, the ∊ subunit was dissociated from F1 during its immobilization and washing before rotation assay (18Nakanishi-Matsui M. Kashiwagi S. Hosokawa H. Cipriano D.J. Dunn S.D. Wada Y. Futai M. J. Biol. Chem. 2006; 281: 4126-4131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The defect of βS174F mutant activity was suppressed by a series of replacements of the βGly-149 residue (26Iwamoto A. Omote H. Hanada H. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 16350-16355Abstract Full Text PDF PubMed Google Scholar, 27Iwamoto A. Park M-Y. Maeda M. Futai M. J. Biol. Chem. 1993; 268: 3156-3160Abstract Full Text PDF PubMed Google Scholar). The single revolution times of the revertant βG149A/βS174F and βG149A/βS174L were similar to that of the wild type. βGly-149 is the first residue of the P-loop containing catalytic residues such as βLys-155 and βThr-156, whereas βSer-174 is located in β-sheet 4 (Fig. 1). The conformation of the β-sheet 4/loop/a-helix B/P-loop domain is strikingly different between the empty (βE) and nucleotide-bound (βDP and βTP) β subunit (8Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2743) Google Scholar) (Fig. 1, b, and c). Thus, the conformational changes of the P-loop during catalysis affect β-sheet 4 through α-helix B for the rotation. Substitution of βSer-174 possibly affected the conformational transition (βD → βE or βE → βT) of the entire domain and increased the pausing dwell. The transition became similar to the wild type with the second mutation, βG149A. It should be important to discuss which rotation step is related to the conformation transition. 120° revolution was observed initially for an actin filament connected to Bacillus F1, when the ATP concentration was lowered (20Yasuda R. Noji H. Kinosita Jr., K. Yoshida M. Cell. 1998; 93: 1117-1124Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar). Using 40-nm gold beads, Yasuda et al. (19Yasuda R. Noji H. Yoshida M. Kinosita Jr., K. Ito H. Nature. 2001; 410: 898-904Crossref PubMed Scopus (705) Google Scholar) further observed 90° and 30° substeps in each 120° step, which were later revised to 80° and 40°, respectively, by the same group (21Shimabukuro K. Yasuda R. Muneyuki E. Hara K.Y. Kinosita Jr., K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14731-14736Crossref PubMed Scopus (214) Google Scholar). The dwell before the 80° revolution was dependent on the ATP concentration, whereas that before the 40° substep was not. These results indicated that the 80° and 40° substeps were driven by ATP binding and hydrolysis/product release, respectively. We assayed rotations with a high ATP concentration and often observed pauses upon 120° revolution possibly at one of the two substeps. As discussed above, they paused not at the ATP waiting dwell but possibly at the ATP hydrolysis/product release dwell. Assuming that the E. coli enzyme has the same two substeps as Bacillus, the mutant F1 paused longer before the 40° stepping. Present results of mutants and revertants indicate that the 40° stepping is driven by the conformation transition of the β-sheet4/loop/a-helix B/P loop domain. Hydrolysis in or product release from the catalytic site including P-loop apparently originates this transition. We occasionally observed long pauses (>0.1 s), which apparently lowered bulk-phase ATPase activity. The revertant βG149A/βS174F and βG149A mutant showed long pauses less often than the wild type. These pauses may be because of the Mg-ADP-inhibited form observed previously using duplex beads (440- and 517-nm diameter) (29Hirono-Hara Y. Noji H. Nishiura M. Muneyuki E. Hara K.Y. Yasuda R. Kinosita Jr., K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13649-13654Crossref PubMed Scopus (159) Google Scholar). The wild-type ATPase activity is sensitive to Mg2+ (31Kanazawa H. Horiuchi Y. Takagi M. Ishino Y. Futai M. J. Biochem. 1980; 88: 695-703Crossref PubMed Scopus (40) Google Scholar), which stabilizes the Mg-ADP-inhibited form (32Hyndman D.J. Milgrom Y.M. Bramhall E.A. Cross R.L. J. Biol. Chem. 1994; 269: 28871-28877Abstract Full Text PDF PubMed Google Scholar, 33Jault J.M. Dou C. Grodsky N.B. Matsui T. Yoshida M. Allison W.S. J. Biol. Chem. 1996; 271: 28818-28824Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). On the other hand, the ATPase activities of βG149A/βS174F and βG149A were less sensitive to Mg2+ than that of the wild type (data not shown), similar to the revertant, βG149S/βS174F (26Iwamoto A. Omote H. Hanada H. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 16350-16355Abstract Full Text PDF PubMed Google Scholar). Thus, the long pauses of the revertant and βG149A occurred rarely because their Mg-ADP-inhibited forms were unstable. These results were consistent with higher steady state ATPase activities of the revertant and βG149A. In this regard mutations in the corresponding domain of Bacillus F1 changed the tendency to generate Mg-ADP-inhibited form (34Masaike T. Mitome N. Noji H. Muneyuki E. Yasuda R. Kinosita Jr., K. Yoshida M. J. Exp. Biol. 2000; 203: 1-8Crossref PubMed Google Scholar). In conclusion, using gold beads but not actin filaments, present studies clearly exhibited stochastic fluctuation of F1 rotation and its defect in β subunit mutants. Mutation/suppression studies revealed that the β-sheet 4/loop/a-helix B/P-loop is an important domain to drive rotation and is at least partially responsible for Mg-ADP inhibition. Further single molecule analysis will provide a new insight in enzyme mechanism (35Lu H.P. Xun L. Xie X.S. Science. 1998; 282: 1877-1882Crossref PubMed Google Scholar). We are grateful for the support from Daiichi Pharmaceutical Co. and Eisai Co. Ltd.
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