Rotational Catalysis of Escherichia coli ATP Synthase F1 Sector

Abstract

A similar approach was taken for mutations in the ␤ subunit key region; consistent with its bulk phase ATPase activities, F 1 with the Ser-174 to Phe substitution (␤S174F) 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 ϳ30-fold higher than the K m value for ATP. A ␤Gly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of ␤S174F. The revertant (␤G149A/␤S174F) 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 ␤G149A/␤S174L. These results indicate that the domain between ␤-sheet 4 (␤Ser-174) and P-loop (␤Gly-149) is important to drive rotation.A ubiquitous ATP synthase (FoF 1 ) synthesizes ATP coupled with an electrochemical proton gradient formed by a respiratory chain (for reviews, see Refs 1-5). FoF 1 consists of a catalytic sector, F 1 (␣ 3 ␤ 3 ␥␦⑀), and a membrane-embedded proton pathway, Fo (ab 2 c 10 ), and can reversibly transport protons coupled with ATP hydrolysis. The ␣ and ␤ subunits form a catalytic hexamer (␣ 3 ␤ 3 ), the central space of which is occupied by the ␥ subunit ␣-helices. ATP is synthesized or hydrolyzed cooperatively at a catalytic site in each ␤ subunit as the binding change mechanism predicts (2). The ␥ subunit rotation in ␣ 3 ␤ 3 has been supported by biochemical studies (1, 6, 7), a crystal structure of the ␣ 3 ␤ 3 ␥ complex (8), and video recorded using an actin filament as a probe (9, 10). Consistent with ATP-dependent proton translocation, a ␥⑀c 10 complex rotated relative to the ␣ 3 ␤ 3 ␦ab 2 in the purified FoF 1 (11-14) or its membrane-bound form (15, 16). The rotation of FoF 1 in liposomes has been revealed by means of single molecule fluorescence resonance energy transfer (17).Counterclockwise rotation of the ␥ subunit has been studied more recently with probes giving low viscous drag such as colloidal gold (14,18,19). The three 120°steps in one revolution of Bacillus F 1 were first observed using an actin filament (20), and later the single 120°step was further subdivided into two substeps (80°and 40°) using gold beads that allow finer observation and analysis (14,19,21). The substeps with larger displacement angles (80°) and smaller substeps (40°) are assigned to ATP binding and hydrolysis/product release steps, respectively (19,21,22). We have observed that the rotation speed of beads attached to the Escherichia coli ␥ subunit varied, reflecting stochastic fluctuations (18,23). Although the average speeds were dependent on the diameter of beads, 40-and 60-nm diameter beads rotated with essentially the same rate (18), suggesting that their rotation speeds were close to that of...

Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of γ-β Subunit Interactions in the Catalytic Dwell

“…In contrast, the pause duration before the 40°rotation step was modulated by the slow hydrolysis rate of ATP␥S 2 or by the catalytic site mutant ␤E190D (in the Bacillus PS3 F 1 ), which was found to significantly increase the length of the catalytic dwell (20). These data together indicate that the dwell before the 40°step is the "catalytic dwell" (20) and defines the order of the substeps during the 120°rotation step observed in high Mg⅐ATP concentrations (21).…”

“…Furthermore, the free Mg 2ϩ concentration is relatively low (Ͻ0.4 mM) to reduce its inhibitory effects (38). The beads occasionally exhibited long pauses (Ͼ0.1 s), which play a role in the bulk steady state ATPase activity (21) (also see below). They were not considered in the present study because we are only analyzing the partial reactions of continuous rotational catalysis, which includes the ratelimiting step.…”

Stochastic rotational catalysis of proton pumping F-ATPase

“…These results suggested that approximately 10% of the F 1 molecules were rotating at the milliseconds time resolution. This interpretation became more convincing on observing longer time courses of randomly selected beads (Nakanishi-Matsui et al His-tag glass When the beads were followed for a longer time, we observed that they unexpectedly paused sometimes for longer than 0.1 s, possibly due to MgADP inhibition (Nakanishi-Matsui et al 2007). Longer pause of single beads should certainly lower the steady-state ATPase, which corresponds to the average rate of randomly selected beads.…”

“…Thus, the rotation pauses before the 808 step, when the ATP concentration was lowered. The 1208 steps observed with a higher ATP concentration most probably reflected ATP hydrolysis/product release (Nakanishi-Matsui et al 2007). …”

“…Gold beads were attached to the mutant g subunit (Nakanishi- Matsui et al 2007), expecting that they reflect rotation as the speed of the g subunit. Similarly, in the case of the wild-type, the single revolution time of a 60 nm gold bead showed stochastic fluctuation.…”