The goal of this study is to characterize the kinetic mechanism of Ca2+ activation and inactivation of cardiac troponin C (cTnC), the Ca2+ signaling protein which triggers heart muscle contraction. Previous studies have shown that IAANS covalently coupled to Cys84 of wild-type cTnC is sensitive to conformational change caused by Ca2+ binding to the regulatory site II; the present study also utilizes the C35S mutant, in which Cys84 is the lone cysteine, to ensure the specificity of IAANS labeling. Site II Ca2+ affinities for cTnC-wt, cTnC-C35S, cTnC-wt-IAANS2, and cTnC-C35S-IAANS were similar (KD = 2-5 microM at 25 degrees C; KD = 2-8 microM at 4 degrees C), indicating that neither the IAANS label nor the C35S mutation strongly perturbs site II Ca2+ affinity. To directly determine the rate of Ca2+ dissociation from site II, the Ca2+-loaded protein was rapidly mixed with a spectroscopically sensitive chelator in a stopped flow spectrometer. The resulting site II Ca2+ off-rates were k(off) = 700-800 s(-1) (4 degrees C) for both cTnC-wt and cTnC-C35S, yielding calculated macroscopic site II Ca2+ on-rates of k(on) = k(off)/KD = 2-4 x 10(8) M(-1) s(-1) (4 degrees C). As observed for Ca2+ affinities, neither the C35S mutation nor IAANS labeling significantly altered the Ca2+ on- and off-rates. Using IAANS fluorescence as a monitor of the protein conformational state, the intramolecular conformational changes (delta) induced by Ca2+ binding and release at site II were found to be significantly slower than the Ca2+ on- and off-rates. The conformational rate constants measured for cTnC-wt-IAANS2 and cTnC-C35S-IAANS were k(delta on) = 120-210 s(-1) and k(delta off) = 90-260 s(-1) (4 degrees C) . Both conformational events were slowed in cTnC-wt-IAANS2 relative to cTnC-C35S-IAANS, presumably due to the bulky IAANS probe coupled to Cys35. Together, the results provide a nearly complete kinetic description of the Ca2+ activation cycle of isolated cTnC, revealing rapid Ca2+ binding and release at site II accompanied by slow conformational steps that are likely to be retained by the full troponin complex during heart muscle contraction and relaxation.