Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase

The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K-d), the enthalpy (Delta H-B) and entropy (T Delta S-B) of binding, and the heat capacity change (Delta C-p). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K-d2',K-5'-ADP = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (Delta C-p) values for 2',5'-ADP (-210 cal mol(-1) K-1), NADP(+) (-230 cal mol(-1) K-1), and H(4)NADP (-220 cal mol(-1) K-1) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TASB = - 13400 +/- 200 cal mol(-1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TASB = -11200 +/- 300 cal mol-1, 35 degrees C indicate that the number of accessible conformational substates of the protein increases upon 2',5'ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional. domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-1), K-d = 37 mu M) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings.