Hsp90 is an ATP-dependent molecular chaperone whose mechanism is not yet understood in detail. Here, we present the first ATPase cycle for the mitochondrial member of the Hsp90 family called Trap1 (tumor necrosis factor receptor-associated protein 1). Using biochemical, thermodynamic, and rapid kinetic methods we dissected the kinetics of the nucleotide-regulated rearrangements between the open and the closed conformations. Surprisingly, upon ATP binding, Trap1 shifts predominantly to the

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... conformation (70%), but, unlike cytosolic Hsp90 from yeast, this process is rather slow at 0.076 s ؊1 . Because reopening (0.034 s ؊1 ) is about ten times faster than hydrolysis (k hyd ‫؍‬ 0.0039 s ؊1 ), which is the rate-limiting step, Trap1 is not able to commit ATP to hydrolysis. The proposed ATPase cycle was further scrutinized by a global fitting procedure that utilizes all relevant experimental data simultaneously. This analysis corroborates our model of a two-step binding mechanism of ATP followed by irreversible ATP hydrolysis and a one-step product (ADP) release. * This work was supported by the Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft (DFG) (Grant RE 1212/1-2 to J. R.), fellowships from the Fonds der chemischen Industrie and the Studienstiftung des Deutschen Volkes (to H. W.), and DFG (Grant SFB 594) and the Fonds der chemischen Industrie (to J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S9. . 3 The abbreviations used are: Hsp90, heat shock protein 90; ITC, isothermal titration calorimetry; Trap1, tumor necrosis factor-associated protein 1; RA, radicicol; (P␥)-MABA-ATP, adenosine triphospho-␥-(NЈmethylanthraniloylaminobutyl)phosphoramidate; AMP-PCP, adenylylmethylene diphosphonat. 4 D. Picard. The web site (www.hsp90.org) provides a comprehensive overview of this research field as well as meetings for the community interested in Hsp90.