CONTRIBUTION OF BACKBONE DYNAMICS TO ENTROPY CHANGES OCCURRING ON OXIDATION OF CYTOCHROME B(5) - CAN REDOX LINKED CHANGES IN HYDROGEN-BOND NETWORKS MODULATE REDUCTION POTENTIALS

Changes in backbone dynamics occurring upon oxidation of rat cytochrom e bs have been examined through model free analyses of N-15-relaxation rates of both oxidation states of the protein. Based on the observed changes, an upper bound for the contribution of backbone dynamics to t he entropy change associated with oxidation has been calculated. The m agnitude of this backbone contribution, 70 +/- 7 J/K.mol, is strikingl y similar to the total entropy change associated with oxidation of the protein determined through an analysis of the temperature dependence of the reduction potential. Origins of the differences in dynamic beha vior of the oxidized and reduced proteins can be attributed to redox l inked changes in hydrogen bond strengths based on large-scale differen ces in amide proton exchange rates observed between the oxidation stat es. Based on these observations the magnitude and possible significanc e of entropic contributions to the electromotive force are discussed. Analysis of the N-15-relaxation rates included modeling of anisotropic diffusional behavior which was expected based on the distinct physica l asymmetry of the protein. An axially symmetric diffusion tensor mode l was found to fit the rotational reorientational properties of the pr otein in both oxidation states. The contribution of paramagnetic relax ation to the N-15-relaxation rates of the oxidized protein was calcula ted based on a set of modified Solomon-Bloembergen equations. The dete rmination of the electronic correlation time of the paramagnetic cente r was based on fits to the proton relaxation rate enhancements of prot ons in close proximity to the paramagnetic center. Analyses of the dyn amic properties of the oxidized cytochrome bs were based on multiple f ield (i.e., 500 and 750 MHz) NMR measurements of N-15 T-1 and T-2 rela xation times.