The enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria. Its structure is bipartite, with a proton-conducting, integral membrane portion, F0, and a peripheral portion, F1. Solubilized F1 is composed of five different subunits, (alpha beta)3 gamma delta epsilon, and is active as an ATPase. The function of F-ATPase is to couple proton translocation through F0 with ATP synthesis in F1 (ref.3). Several lines of evidence support the spontaneous formation of ATP on F1 (refs 4,5) and its endergonic release at cooperative and rotating (or at least alternating) sites. The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F0 into F1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)3, the catalytic hexagon of F1 as suggested by electron microscopy, by X-ray crystal structure analysis and by the use of cleavable crosslinkers. Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled gamma. We observe the rotation of gamma relative to immobilized (alpha beta)3 in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F1. Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis, with gamma acting as a crankshaft in (alpha beta)3. The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.

ATP synthase mediates proton flow through its membrane portion, F 0 , which drives the synthesis of ATP in its headpiece, F 1 . The F 1 -portion contains a hexagonal array of three subunits ␣ and three ␤ encircling a central subunit ␥, that in turn interacts with a smaller and with F 0 . Recently we reported that the application of polarized absorption recovery after photobleaching showed the ATP-driven rotation of ␥ over at least two, if not three, ␤. Here we extend probes of such rotation aided by a new theory for assessing continuous versus stepped, Brownian versus unidirectional molecular motion. The observed relaxation of the absorption anisotropy is fully compatible with a unidirectional and stepping rotation of ␥ over three equidistantly spaced angular positions in the hexagon formed by the alternating subunits ␣ and ␤. The results strongly support a rotational catalysis with equal participation of all three catalytic sites. In addition we report a limited rotation of ␥ without added nucleotides, perhaps idling and of Brownian nature, that covers only a narrow angular domain.

No abstract

Nature invented molecular rotatory devices such as the f lagellar motor and ATP synthase. Photoselection techniques have been frequently used to detect the rotational random walk of proteins but only rarely for the rotational drift of subunits in proteins. Pertinent theories predict an oscillatory behavior of the polarization anisotropy, r, for unidirectional rotational drift, as opposed to a monotonic relaxation of r for bidirectional random walk. The underlying assumption of an angular continuum is questionable for intersubunit rotation in proteins. We developed a theory for stepped rotatory devices. It predicts the damped oscillation of r under unidirectional drift. Damping increases with decreasing number of steps. For only three steps a quasi-monotonic relaxation of r is predicted for both random walk and drift. In photoselection experiments with active F-ATPase we observed the relaxation of r when a spectroscopic probe was attached to the central ␥-subunit. This behavior is compatible with the expectation for a three-stepped rotatory device.In the flagellar motor and in ATP synthase (F-ATPase) protein subunits rotate against each other. The rotation of bacterial flagellae has been detected in the light microscope by the rotation of the cell body after tethering of the flagellae to a solid support (1). The detection of rotatory motion in objects of smaller size like subunits in ATP-synthase or the load-free flagellar motor requires less macroscopic techniques. Recently, we detected and time resolved the activity-linked intersubunit rotation in F-ATPase by PARAP (polarized absorption recovery after photobleaching) (2). This spectroscopic technique applies to a large ensemble of molecules and is based on orientational photoselection. Rotational motion causes a transient of the polarization anisotropy, r (see Eq. 1). In these studies we observed a monotonic relaxation of the polarization anisotropy of a probe which was attached to ␥-subunit of this enzyme. It occurred in about 100 ms, which conformed with the turnover time of the enzyme under the given experimental conditions (see Fig. 4A for data and Fig. 3 for the enzyme structure).Does the transient behavior of r allow to discriminate between rotational random walk which is bidirectional from rotational drift under a driving force which is unidirectional? For a true rotatory motor-i.e., for drift without superimposed random walk-one expects an oscillatory transient of the r-parameter, and for rotatory random walk a monotonic decay. Theories for rotatory random walk and drift have been published by various authors (3-7). A theory by Hoshikawa and Asai (7) supports the above-mentioned expectation. Applying their equation 23 to a situation where the stator of a molecular rotatory motor is immobilized and a spectroscopic probe is attached to the rotor, an undamped oscillation of r is predicted.Their approach, however, is based on Fick's phenomenological equation for diffusion in a continuous angular space. Considering the flagellar motor and even more ...