**Effect of the substrate concentration dependence on the observed rate constant ***k*_{2(obs)} measured at 4 °C in laser photoexcitation experiments. The reaction mixture contained 30 μm NiR, 200 μm NADH. and 50 mm NMN in 50 mm KP_{i}. In a control experiment, the total ionic strength was adjusted to [KNO_{2}] + [KCl] = 50 mm (*open squares* in *A* and *B*), to ascertain that the changing ion concentration upon titration of nitrite was negligible. The *error bars* are smaller than the symbol size if invisible. *A* and *B*, *closed squares*, 100% H_{2}O, pH 7.0; *open squares*, data obtained in 100% H_{2}O with an ionic strength of [KNO_{2}] + [KCl] = 50 mm; *solid lines*, fit to an equation reflecting two substrate-binding sites (, 100% H_{2}O, pH 7.0); *dotted lines*, fit to D_{2}O data set shown in *C* and *D. C* and *D*, *open circles*, 95% D_{2}O, pD 7.0; *solid lines*, fit to a hyperbolic equation (95% D_{2}O, pD 7.0, ); *dotted lines*, fit to H_{2}O data set shown in *A* and *B. E* and *F*, substrate concentration dependence of the SKIE obtained as the ratio of the observed rate constants *k*_{2(obs)} in H_{2}O and D_{2}O. The *solid line* represents a fit to a simplified equation reflecting two substrate-binding sites (). *G*, proton inventory plots. The ratio of the observed rate constant *k*_{2(obs)},_{n}, obtained at a certain volume fraction of D_{2}O(*n*), and the rate constant *k*_{2(obs), 0} in pure H_{2}O is plotted *versus n*. The pH values of D_{2}O-containing buffers were adjusted according to . *Squares*, substrate-free sample; *circles*, sample mixture including 10 mm KNO_{2}. The *solid lines* are fits to . Please note that the data sets presented in *A* and *B* (*C* and *D*; *E* and *F*) are identical. The data in *A* (*C*; *E*) are plotted logarithmically over the complete concentration range, whereas the data in *B* (*D*; *F*) are shown over a linear concentration range. To ensure the recognizability of the behavior at lower substrate concentrations on the linear scale (*B*; *D*), the presented concentration range is slightly smaller than on the logarithmic plot (*A*; *C*).

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