Figure 5.

The kinetics of reduction of CO-bound CYP450 by CPR in the presence of benzphetamine at different temperatures (11 – 38 °C). (a) Soret-band region in the UV spectra of CO-bound CYP450 2B4 showing a decrease in the intensity of the peak at 417 nm and the appearance of a new peak at 451.25 nm (indicated with arrows). (b) Temperature-dependent kinetic traces (@451.25 nm) from the reduced (Fe²⁺) CO-bound CYP450 2B4 in the presence of CPR and benzphetamine. (c) Redox reaction rate constants at different temperatures. The straight-line shows a non-linear change of k1 with temperature. (d) Contribution of the slow and the rapid phases of reactions calculated using their amplitudes (a1 and a2) at different temperatures. The samples used in stopped-flow experiments were prepared in a 10 mM Tris buffer (pH 7.4) containing 50 mM NaCl. After mixing, the sample consisted of 1.5 μM proteins, as well as 10 and 20 molar equivalents of benzphetamine and NADPH, respectively.