Abstract:
:A combination of mutagenesis, calorimetry, kinetics, and small-angle X-ray scattering (SAXS) has been used to study the mechanism of ligand binding energy propagation through human cytochrome P450 reductase (CPR). Remarkably, the energetics of 2',5'-ADP binding to R597 at the FAD-binding domain are affected by mutations taking place at an interdomain loop located 60 A away. Either deletion of a 7 amino acid long segment (T236-G237-E238-E239-S240-S241-I242) or its replacement by poly-proline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding (DeltaHB). This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-CPR complex. Moreover, the estimated heat capacity change (DeltaCp) for this interaction changes from -220 cal mol-1 K-1 in the wild-type enzyme to -580 cal mol-1 K-1 in the deletion mutant. Pre-steady-state kinetics measurements reveal a 50-fold decrease in the microscopic rate for interdomain (FAD --> FMN) electron transfer in the deletion mutant (kobs = 0.4 s-1). Multiple turnover cytochome c reduction assays indicate that these mutations impair the ability of the FMN-binding domain to shuttle electrons from the FAD-binding domain to the cytochrome partner. Binding of 2',5'-ADP to wild-type CPR triggers a large-scale structural rearrangement resulting in the complex having a more compact domain organization, and the maximum molecular dimension (Dmax) decreases from 110 A in ligand-free enzyme to 100 A in the ligand-bound CPR. The SAXS experiments also demonstrate that what is affected by the mutations is indeed the relative diffusional motion of the domains. Furthemore, ab initio shape reconstruction and homology modeling would suggest that-in the deletion mutant-hindering of domain motion occurs concomitantly with dimerization. The results presented here show that the energetics of this highly localized interaction (2',5'-ADP binding) have a global character, and are highly sensitive to functional structural dynamics involving distal domains. These findings support early theoretical studies which postulate a single protein molecule to be a real, independent thermodynamic ensemble.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Grunau A,Geraki K,Grossmann JG,Gutierrez Adoi
10.1021/bi700596ssubject
Has Abstractpub_date
2007-07-17 00:00:00pages
8244-55issue
28eissn
0006-2960issn
1520-4995journal_volume
46pub_type
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